Phosphate Headgroup Research Articles

Hydration dynamics of a lipid membrane: Hydrogen bond networks and lipid-lipid associations

Dynamics of hydration layers of a dimyristoylphosphatidylcholine (DMPC) bilayer are investigated using an all atom molecular dynamics simulation. Based upon the geometric criteria, continuously residing interface water molecules which form hydrogen bonds solely among themselves and then concertedly hydrogen bonded to carbonyl, phosphate, and glycerol head groups of DMPC are identified. The interface water hydrogen bonded to lipids shows slower relaxation rates for translational and rotational dynamics compared to that of the bulk water and is found to follow sub-diffusive and non-diffusive behaviors, respectively. The mean square displacements and the reorientational auto-correlation functions are slowest for the interfacial waters hydrogen bonded to the carbonyl oxygen since these are buried deep in the hydrophobic core among all interfacial water studied. The intermittent hydrogen bond auto-correlation functions are calculated, which allows breaking and reformations of the hydrogen bonds. The auto-correlation functions for interfacial hydrogen bonded networks develop humps during a transition from cage-like motion to eventual power law behavior of t−3/2. The asymptotic t−3/2 behavior indicates translational diffusion dictated dynamics during hydrogen bond breaking and formation irrespective of the nature of the chemical confinement. Employing reactive flux correlation analysis, the forward rate constant of hydrogen bond breaking and formation is calculated which is used to obtain Gibbs energy of activation of the hydrogen bond breaking. The relaxation rates of the networks buried in the hydrophobic core are slower than the networks near the lipid-water interface which is again slower than bulk due to the higher Gibbs energy of activation. Since hydrogen bond breakage follows a translational diffusion dictated mechanism, chemically confined hydrogen bond networks need an activation energy to diffuse through water depleted hydrophobic environments. Our calculations reveal that the slow relaxation rates of interfacial waters in the vicinity of lipids are originated from the chemical confinement of concerted hydrogen bond networks. The analysis suggests that the networks in the hydration layer of membranes dynamically facilitate the water mediated lipid-lipid associations which can provide insights on the thermodynamic stability of soft interfaces relevant to biological systems in the future.

Interfacial water molecules at biological membranes: Structural features and role for lateral proton diffusion.

Proton transport at water/membrane interfaces plays a fundamental role for a myriad of bioenergetic processes. Here we have performed ab initio molecular dynamics simulations of proton transfer along two phosphatidylcholine bilayers. As found in previous theoretical studies, the excess proton is preferably located at the water/membrane interface. Further, our simulations indicate that it interacts not only with phosphate head groups, but also with water molecules at the interfaces. Interfacial water molecules turn out to be oriented relative to the lipid bilayers, consistently with experimental evidence. Hence, the specific water-proton interaction may help explain the proton mobility experimentally observed at the membrane interface.

Biophysical Studies Reveal Key Interactions between Papiliocin-Derived PapN and Lipopolysaccharide in Gram-Negative Bacteria.

Papiliocin, isolated from the swallowtail butterfly (Papilio xuthus), is an antimicrobial peptide with high selectivity against gram-negative bacteria. We previously showed that the N-terminal helix of papiliocin (PapN) plays a key role in the antibacterial and anti-inflammatory activity of papiliocin. In this study, we measured the selectivity of PapN against multidrug-resistant gram-negative bacteria, as well as its anti-inflammatory activity. Interactions between Trp2 of PapN and lipopolysaccharide (LPS), which is a major component of the outer membrane of gram-negative bacteria, were studied using the Trp fluorescence blue shift and quenching in LPS micelles. Furthermore, using circular dichroism, we investigated the interactions between PapN and LPS, showing that LPS plays critical roles in peptide folding. Our results demonstrated that Trp2 in PapN was buried deep in the negatively charged LPS, and Trp2 induced the α-helical structure of PapN. Importantly, docking studies determined that predominant electrostatic interactions of positively charged arginine residues in PapN with phosphate head groups of LPS were key factors for binding. Similarly, hydrophobic interactions by aromatic residues of PapN with fatty acid chains in LPS were also significant for binding. These results may facilitate the development of peptide antibiotics with anti-inflammatory activity.

The effect of chitin nanoparticles on surface behavior of DPPC/DPPG Langmuir monolayers

The effect of chitin nanoparticles on surface behavior of lipid systems containing dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) is studied by surface pressure (π)-area (A) isotherms, polarization-modulation infrared reflection absorption spectroscopy (PM-IRRAS), Brewster angle microscopy (BAM). The variation of surface behavior of DPPC/DPPG monolayers is induced mainly by electrostatic interactions between nanoparticles and head groups of phospholipids. At lower surface pressure, nanoparticles can penetrate into the monolayers and the positive charges carried by nanoparticles benefits the enrichment of phospholipid molecules at surface, which not only increases the mean molecular area but also hinders the formation of phospholipid liquid-condensed (LC) phase. However, when surface pressure is higher, the nanoparticles flee away from the surface and some of the phospholipid molecules are pulled out of the monolayers together to the subphase and decrease the order degree of the monolayers. Moreover, nanoparticles can destroy the hydrogen-bond between water molecules and phosphate head groups and thus lead to the dehydration of phosphate groups. This work confirms that chitin nanoparticles can affect the surface behavior of DPPC/DPPG monolayers. Furthermore, the results obtained using mixed monolayer containing two major lung surfactants DPPC/DPPG and nanoparticles will be helpful for deep understanding the harm of PM2.5 to lung health.

Effect of pH and Salt on Surface pKa of Phosphatidic Acid Monolayers.

The pH-induced surface speciation of organic surfactants such as fatty acids and phospholipids in monolayers and coatings is considered to be an important factor controlling their interfacial organization and properties. Yet, correctly predicting the surface speciation requires the determination of the surface dissociation constants (surface pKa) of the protic functional group(s) present. Here, we use three independent methods-compression isotherms, surface tension pH titration, and infrared reflection-absorption spectroscopy (IRRAS)-to study the protonation state of dipalmitoylphosphatidic acid (DPPA) monolayers on water and NaCl solutions. By examining the molecular area expansion at basic pH, the pKa to remove the second proton of DPPA (surface pKa2) at the aqueous interface is estimated. In addition, utilizing IRRAS combined with density functional theory calculations, the vibrational modes of the phosphate headgroup were directly probed and assigned to understand DPPA charge speciation with increasing pH. We find that all three experimental techniques give consistent surface pKa2 values in good agreement with each other. Results show that a condensed DPPA monolayer has a surface pKa2 of 11.5, a value higher than previously reported (∼7.9-8.5). This surface pKa2 was further altered by the presence of Na+ cations in the aqueous subphase, which reduced the surface pKa2 from 11.5 to 10.5. It was also found that the surface pKa2 value of DPPA is modulated by the packing density (i.e., the surface charge density) of the monolayer, with a surface pKa2 as low as 9.2 for DPPA monolayers in the two-dimensional gaseous phase over NaCl solutions. The experimentally determined surface pKa2 values are also found to be in agreement with those predicted by Gouy-Chapman theory, validating these methods and proving that surface charge density is the driving factor behind changes to the surface pKa2.

Zwitterionic and Charged Lipids Form Remarkably Different Structures on Nanoscale Oil Droplets in Aqueous Solution

The molecular structure of zwitterionic and charged monolayers on small oil droplets in aqueous solutions is determined using a combined second harmonic and sum frequency study. From the interfacial vibrational signature of the acyl chains and phosphate headgroups as well as the response of the hydrating water, we find that zwitterionic and charged lipids with identical acyl chains form remarkably different monolayers. Zwitterionic phospholipids form a closely packed monolayer with highly ordered acyl tails. In contrast, the charged phospholipids form a monolayer with a low number density and disordered acyl tails. The charged headgroups are oriented perpendicular to the monolayer rather than parallel, as is the case for zwitterionic lipids. These significant differences between the two types of phospholipids indicate important roles of phospholipid headgroups in the determination of properties of cellular membranes and lipid droplets. The observed behavior of charged phospholipids is different from expectations based on studies performed on extended planar interfaces, at which condensed monolayers are readily formed. The difference can be explained by nanoscale related changes in charge condensation behavior that has its origin in a different balance of interfacial intermolecular interactions.

Effect of temperature, water content and free fatty acid on reverse micelle formation of phospholipids in vegetable oil

The self-assembly of phospholipids in oil, specifically lecithin in rapeseed oil, was investigated by combining experimental and computational methods The influence of temperature, water, and free fatty acids on the onset of lecithin aggregation in the rapeseed oil was determined using the 7,7,8,8 -tetracyanoquinodimethane dye (TCNQ) solubilization method and the size and shape of the self-assembled lecithin structures were investigated by small-angle X-ray scattering and cryogenic transmission electron microscopy. In the absence of excess water in the system (0.03wt-% water in oil), stable cylindrical lecithin reverse micelles were observed above the critical micelle concentration (CMC). Comparing the aggregation response in room temperature and at 70°C revealed that CMC decreased with increasing temperature. Furthermore, already a modest amount of added water (0.3wt-% water in oil) was sufficient to induce the formation of lamellar lecithin structures, that phase separated from the oil. In low water content, oleic acid suppressed the formation of lecithin reverse micelles whereas in the presence of more water, the oleic acid stabilized the reverse micelles. Consequently, more water was needed to induce phase separation in the presence of oleic acid. Molecular dynamics simulations indicated that the stabilizing effect of oleic acid resulted from oleic acid enhancing phospholipid solubilization in the oil by forming a solvating shell around the phosphate head group. The findings showed that the response of the mixed surfactant system is a delicate interplay of the different components and variables. The significance of the observations is that multiple parameters need to be controlled for desired system response, for example towards vegetable oil purification or phospholipid based microemulsions.

Structural insights into ligand recognition by the lysophosphatidic acid receptor LPA6.

Lysophosphatidic acid (LPA) is a bioactive lipid composed of a phosphate group, a glycerol backbone, and a single acyl chain that varies in length and saturation. LPA activates six class A G-protein-coupled receptors to provoke various cellular reactions. Because LPA signalling has been implicated in cancer and fibrosis, the LPA receptors are regarded as promising drug targets. The six LPA receptors are subdivided into the endothelial differentiation gene (EDG) family (LPA1-LPA3) and the phylogenetically distant non-EDG family (LPA4-LPA6). The structure of LPA1 has enhanced our understanding of the EDG family of LPA receptors. By contrast, the functional and pharmacological characteristics of the non-EDG family of LPA receptors have remained unknown, owing to the lack of structural information. Although the non-EDG LPA receptors share sequence similarity with the P2Y family of nucleotide receptors, the LPA recognition mechanism cannot be deduced from the P2Y1 and P2Y12 structures because of the large differences in the chemical structures of their ligands. Here we determine the 3.2 Å crystal structure of LPA6, the gene deletion of which is responsible for congenital hair loss, to clarify the ligand recognition mechanism of the non-EDG family of LPA receptors. Notably, the ligand-binding pocket of LPA6 is laterally open towards the membrane, and the acyl chain of the lipid used for the crystallization is bound within this pocket, indicating the binding mode of the LPA acyl chain. Docking and mutagenesis analyses also indicated that the conserved positively charged residues within the central cavity recognize the phosphate head group of LPA by inducing an inward shift of transmembrane helices 6 and 7, suggesting that the receptor activation is triggered by this conformational rearrangement.

Characterization of 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[Methoxy(polyethylene glycerol)-2000] and Its Complex with Doxorubicin Using Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics.

Polyethylene glycol (PEG) lipid nanoparticles (LNPs) spontaneously assemble in water, forming uniformly sized nanoparticles incorporating drugs with prolonged blood clearance compared to drugs alone. Previously, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycerol)-2000] (DSPE-PEG2000) and several drug adducts, including doxorubicin, were analyzed by a combination of physical and molecular dynamic (MD) studies. In this study, a complete chemical shift assignment of DSPE-PEG2000 plus or minus doxorubicin was achieved using nuclear magnetic resonance (NMR), one-dimensional selective nuclear Overhauser spectroscopy (1D-selNOESY), NOESY, correlation spectroscopy (COSY), total correlated spectroscopy (TOCSY), heteronuclear single quantum coherence (HSQC), and HSQC-TOCSY. Chemical shift perturbation, titration, relaxation enhancement, and NOESY analysis combined with MD reveal detailed structural information at the atomic level, including the location of doxorubicin in the micelle, its binding constant, the hydrophilic shell organization, and the mobility of the PEG2000 tail, demonstrating that NMR spectroscopy can characterize drug-DSPE-PEG2000 micelles with molecular weights above 180 kDa. The MD study revealed that an initial spherical organization led to a more-disorganized oblate structure in an aqueous environment and agreed with the NMR study in the details of the fine structure, in which methyl group(s) of the stearic acid in the hydrophobic core of the micelle are in contact with the phosphate headgroup of the lipid. Although the molecular size of the LNP drug complex is about 180 kDa, atomic resolution can be achieved by NMR-based methods that reveal distinct features of the drug-lipid interactions. Because many drugs have unfavorable blood clearance that may benefit from incorporation into LNPs, a thorough knowledge of their physical and chemical properties is essential to moving them into a clinical setting. This study provides an advanced basic approach that can be used to study a wide range of drug-LNP interactions.

A membrane-spanning macrocyclic bolaamphiphile lipid mimic of archaeal lipids

The synthesis of a 72-membered macrocyclic tetraester bolaamphiphile is accomplished in six chemical steps from commercially available starting materials using copper-accelerated azide–alkyne coupling to close the macrocycle in high yield. Related diester amphiphiles and an acyclic tetraester bolaamphiphile were also prepared. The set of lipids bearing nitrophenyl phosphate head groups were incorporated into phospholipid vesicles but failed to undergo phosphate hydrolysis in basic conditions, undergoing efficient elimination in competition. The same lipid cores bearing phosphate-linked nitrobenzoxadiazole (NBD) head groups also incorporated into phospholipid vesicles and the NBD fluorescence was quenched with cobalt ions. The proportion of membrane-spanning bolaamphiphiles was determined from the ratio of cobalt quenching in the presence and in the absence of a detergent. The macrocyclic bolaamphiphile is incorporated into phospholipid vesicles such that 48 ± 4% of the NBD head groups are in the outer leaflet, consistent with a membrane-spanning orientation. The acyclic bolaamphiphile is incorporated with 75 ± 3% of the NBD head groups accessible to quencher in the absence of a detergent suggesting U-shaped incorporation in the outer leaflet of the bilayer membrane. In ring size and spanning ability, the macrocyclic bolaamphiphile mimics naturally occurring macrocyclic archaeal lipids.

Conformational Space of a Polyphilic Molecule with a Fluorophilic Side Chain Integrated in a DPPC Bilayer.

We investigate the conformational space of a polyphilic molecule with hydrophilic, lipophilic and fluorophilic parts inserted as a transmembrane agent into a dipalmitoylphosphatidylcholine bilayer by means of all-atom molecular dynamics simulations. Special focus is put on the competing structural driving forces arising from the hydrophilic, lipophilic and fluorophilic side chains and the aromatic backbone of the polyphile. We observe a significant difference between the lipophilic and the fluorophilic side chains regarding their intramembrane distribution. While the lipophilic groups remain membrane-centered, the fluorophilic parts tend to orient toward the phosphate headgroups. This trend is important for understanding the influence of polyphile agents on the properties of phospholipid membranes. From a fundamental point of view, our computed distribution functions of the side chains are related to the interplay of sterical, enthalpic and entropic driving forces. Our findings illustrate the potential of rationally designed membrane additives which can be exploited to tune the properties of phospholipid membranes. © 2017 Wiley Periodicals, Inc.

Lipid Protein Interaction Mediates Ion Transport in P2X Receptors

ATP a ubiquitously prevalent biomolecule, apart from being the principal energetic source for living cells, also plays a crucial role in inter-cellular communication, acting as a signaling molecule. One of the major receptors in this signaling cascade are the P2X receptors which are are trimeric, non-selective cation channel activated by ATP and are responsible for key processes such as muscle contraction, neurotransmission, inflammatory response, pain, and taste signal transduction. As a result of their extensive prevalence and important implications in human physiology, P2X receptors serve as important pharmacological targets for cardiovascular, neuronal, and inflammatory diseases. Recently our collaborator Dr Eric Gouax was successful in crystallizing full P2X (human P2X3), which provided us an unprecedented opportunity to explore different facets of ion permeation mechanism and pathway exploration in a lipid rich environment. We have performed an all atom MD simulation of a full P2X channel, embedded in a POPC bi-layer. In order to accelerate the ion permeation, process a constant electric field of 1 V was applied along the membrane normal. Upon the application of external electric field we observed multiple ion permeation events, with the ion entering through the extracellular fenestrations And exiting through the lateral fenestrations that was deeply embedded in the membrane. Interestingly the ion permeation pathway was lined with the lipid head groups which had entered through the lateral fenestrations. The negatively charged phosphate head groups of the POPC lipids interacts with the Sodium ion and assisted in the permeation of the ion through the pore. Thus through this work we were able to propose a unique ion permeation architecture for P2X receptors where the pathway was made of both protein and lipids.

Free-radicals and advanced chemistries involved in cell membrane organization influence oxygen diffusion and pathology treatment.

A breakthrough has been discovered in pathology chemistry related to increasing molecular structure that can interfere with oxygen diffusion through cell membranes. Free radicals can crosslink unsaturated low-viscosity fatty acid oils by chain-growth polymerization into more viscous liquids and even solids. Free radicals are released by mitochondria in response to intermittent hypoxia that can increase membrane molecular organization to reduce fluidity and oxygen diffusion in a possible continuing vicious cycle toward pathological disease. Alternate computational chemistry demonstrates molecular bond dynamics in free energy for cell membrane physiologic movements. Paired electrons in oxygen and nitrogen atoms require that oxygen bonds rotate and nitrogen bonds invert to seek polar nano-environments and hide from nonpolar nano-environments thus creating fluctuating instability at a nonpolar membrane and polar biologic fluid interface. Subsequent mechanomolecular movements provide free energy to increase diffusion by membrane transport of molecules and oxygen into the cell, cell-membrane signaling/recognition/defense in addition to protein movements for enzyme mixing. In other chemistry calcium bonds to membrane phosphates primarily on the outer plasma cell membrane surface to influence the membrane firing threshold for excitability and better seal out water permeation. Because calcium is an excellent metal conductor and membrane phosphate headgroups form a semiconductor at the biologic fluid interface, excess electrons released by mitochondria may have more broad dissipation potential by safe conduction through calcium atomic-sized circuits on the outer membrane surface. Regarding medical conditions, free radicals are known to produce pathology especially in age-related disease in addition to aging. Because cancer cell membranes develop extreme polymorphism that has been extensively followed in research, accentuated easily-visualized free-radical models are developed. In terms of treatment, use of vitamin nutrient supplements purported to be antioxidants that remove free radicals has not proved worthwhile in clinical trials presumably due to errors with early antioxidant measurements based on inaccurate colorimetry tests. However, newer covalent-bond shrinkage tests now provide accurate measurements for free-radical inhibitor hydroquinone and other molecules toward drug therapy.

On the Structural and Dynamical Properties of DOPC Reverse Micelles.

The structure and dynamics of phospholipid reverse micelles are studied by molecular dynamics. We report all-atom unconstrained simulations of 1,2-dioleoyl-sn-phosphatidylcholine (DOPC) reverse micelles in benzene of increasing sizes, with water-to-surfactant number ratios ranging from W0 = 1 to 16. The aggregation number, i.e., the number of DOPC molecules per reverse micelle, is determined to fit experimental light-scattering measurements of the reverse micelle diameter. The simulated reverse micelles are found to be approximately spherical. Larger reverse micelles (W0 > 4) exhibit a layered structure with a water core and the hydration structure of DOPC phosphate head groups is similar to that found in phospholipid membranes. In contrast, the structure of smaller reverse micelles (W0 ≤ 4) cannot be described as a series of concentric layers successively containing water, surfactant head groups, and surfactant tails, and the head groups are only partly hydrated and frequently present in the core. The dynamics of water molecules within the phospholipid reverse micelles slow down as the reverse micelle size decreases, in agreement with prior studies on AOT and Igepal reverse micelles. However, the average water reorientation dynamics in DOPC reverse micelles is found to be much slower than in AOT and Igepal reverse micelles with the same W0 ratio. This is explained by the smaller water pool and by the stronger interactions between water and the charged head groups, as confirmed by the red-shift of the computed infrared line shape with decreasing W0.

Synthesis of substrates for periodate-coupled assay of phospholipases C and sphingomyelinases.

A series of 4-nitrophenyl (pNP) and 4-methylumbelliferyl (4MU) substrate analogues of phosphatidyl choline (PC) and phosphatidic acid (PA) were synthesized from 4-bromo-1-butene by ether formation, olefin epoxidation and ring opening with the phosphate head group. The pNP PC analogue, 4-(4-nitrophenoxy)-2-hydroxy-butyl-1-phosphoryl choline (1) was evaluated in assays of fungal sphingomyelinases, also displaying phospholipase C activity. Reactions were terminated with a periodate-containing stop solution, leading to liberation of pNP, quantified spectrophotometrically in an end-point measurement. A kinetic evaluation of sphingomyelinases from Kionochaeta sp. and Penicillium emersonii showed relatively high KM and low kcat values for this substrate, limiting its practical applicability in assays with low sphingomyelinase concentrations.

Structural Insights into the Transport Mechanism of the Human Sodium-dependent Lysophosphatidylcholine Transporter MFSD2A

Major facilitator superfamily domain containing 2A (MFSD2A) was recently characterized as a sodium-dependent lysophosphatidylcholine transporter expressed at the blood-brain barrier endothelium. It is the primary route for importation of docosohexaenoic acid and other long-chain fatty acids into fetal and adult brain and is essential for mouse and human brain growth and function. Remarkably, MFSD2A is the first identified major facilitator superfamily member that uniquely transports lipids, implying that MFSD2A harbors unique structural features and transport mechanism. Here, we present three three-dimensional structural models of human MFSD2A derived by homology modeling using MelB- and LacY-based crystal structures and refined by biochemical analysis. All models revealed 12 transmembrane helices and connecting loops and represented the partially outward-open, outward-partially occluded, and inward-open states of the transport cycle. In addition to a conserved sodium-binding site, three unique structural features were identified as follows: a phosphate headgroup binding site, a hydrophobic cleft to accommodate a hydrophobic hydrocarbon tail, and three sets of ionic locks that stabilize the outward-open conformation. Ligand docking studies and biochemical assays identified Lys-436 as a key residue for transport. It is seen forming a salt bridge with the negative charge on the phosphate headgroup. Importantly, MFSD2A transported structurally related acylcarnitines but not a lysolipid without a negative charge, demonstrating the necessity of a negatively charged headgroup interaction with Lys-436 for transport. These findings support a novel transport mechanism by which lysophosphatidylcholines are "flipped" within the transporter cavity by pivoting about Lys-436 leading to net transport from the outer to the inner leaflet of the plasma membrane.

The Localization of Phenolic Compounds in Liposomal Bilayers and Their Effects on Surface Characteristics and Colloidal Stability.

The interactions with and effects of five chemically distinct, bioactive phenolic compounds on the lipid bilayers of model dipalmitoylphosphatidylcholine (DPPC) liposomes were investigated. Complementary analytical techniques, including differential scanning calorimetry (DSC) and phosphorus and proton nuclear magnetic resonance spectroscopy (NMR), were employed in order to determine the location of the compounds within the bilayer and to correlate location with their effects on bilayer characteristics and liposomal stability. As compared to the phenolic compounds localized in the glycerol region of the DPPC head group within the bilayer, which enhanced the colloidal stability of the liposomes, compounds located closer to the center of the bilayer reduced vesicle stability as a function of time. Molecules present in the upper region of liposomal DPPC acyl chains (C1-C10) inhibited liposomal aggregation and size increase, perhaps due to tighter packing of adjoining DPPC molecules and increased surface exposure of DPPC phosphate head groups. These data may be useful for designing liposomal systems containing hydrophobic phenols and other small molecules, selecting appropriate analytical methods for determining their location within liposomal bilayers, and predicting their effects on liposome characteristics early in the liposome formulation development process.

Male functions and malfunctions: the impact of phosphoinositides on pollen development and pollen tube growth.

Phosphoinositides in pollen. In angiosperms, sexual reproduction is a series of complex biological events that facilitate the distribution of male generative cells for double fertilization. Angiosperms have no motile gametes, and the distribution units of generative cells are pollen grains, passively mobile desiccated structures, capable of delivering genetic material to compatible flowers over long distances and in an adverse environment. The development of pollen (male gametogenesis) and the formation of a pollen tube after a pollen grain has reached a compatible flower (pollen tube growth) are important aspects of plant developmental biology. In recent years, a wealth of information has been gathered about the molecular control of cell polarity, membrane trafficking and cytoskeletal dynamics underlying these developmental processes. In particular, it has been found that regulatory membrane phospholipids, such as phosphoinositides (PIs), are critical regulatory players, controlling key steps of trafficking and polarization. Characteristic features of PIs are the inositol phosphate headgroups of the lipids, which protrude from the cytosolic surfaces of membranes, enabling specific binding and recruitment of numerous protein partners containing specific PI-binding domains. Such recruitment is globally an early event in polarization processes of eukaryotic cells and also of key importance to pollen development and tube growth. Additionally, PIs serve as precursors of other signaling factors with importance to male gametogenesis. This review highlights the recent advances about the roles of PIs in pollen development and pollen function.

Solvation of Calcium-Phosphate Headgroup Complexes at the DPPC/Aqueous Interface.

The effects of sodium (Na(+) ) and calcium (Ca(2+) ) cations on model zwitterionic dipalmitoylphosphatidylcholine (DPPC) monolayers spread on metal chloride salt solutions are investigated by means of vibrational sum frequency generation (VSFG) and heterodyne-detected (HD)-VSFG spectroscopy. VSFG and HD-VSFG spectra in the OH stretching region reveal cation-specific effects on the interfacial water's H-bonding network, knowledge of which has been limited to date. It is found that low-concentrated Ca(2+) more strongly perturbs interfacial water organization relative to highly concentrated Na(+) . At higher Ca(2+) concentrations, the water H-bonding network at the DPPC/CaCl2 interface reorganizes and the resulting spectrum closely follows that of the bare air/CaCl2 interface up to ∼3400 cm(-1) . Most interesting is the appearance of a negative band at ∼3450 cm(-1) in the DPPC/CaCl2 Im χs ((2)) spectra, likely arising from an asymmetric solvation of Ca(2+) -phosphate headgroup complexes. This gives rise to an electric field that orients the net OH transition moments of a subset of OH dipoles toward the bulk solution.

Radio Frequency-Activated Nanoliposomes for Controlled Combination Drug Delivery.

This work was conducted in order to design, characterize, and evaluate stable liposomes containing the hydrophobic drug raloxifene HCl (RAL) and hydrophilic doxycycline HCl (DOX), two potentially synergistic agents for treating osteoporosis and other bone lesions, in conjunction with a radio frequency-induced, hydrophobic magnetic nanoparticle-dependent triggering mechanism for drug release. Both drugs were successfully incorporated into liposomes by lipid film hydration, although combination drug loading compromised liposome stability. Liposome stability was improved by reducing the drug load and by including Pluronics® (PL) in the formulations. DOX did not appear to interact with the phospholipid membranes comprising the liposomes, and its release was maximized in the presence of radio frequency (RF) heating. In contrast, differential scanning calorimetry (DSC) and phosphorus-31 nuclear magnetic resonance ((31)P-NMR) analysis revealed that RAL developed strong interactions with the phospholipid membranes, most notably with lipid phosphate head groups, resulting in significant changes in membrane thermodynamics. Likewise, RAL release from liposomes was minimal, even in the presence of RF heating. These studies may offer useful insights into the design and optimization of multidrug containing liposomes. The effects of RAL on liposome characteristics and drug release performance underscore the importance of appropriate physical-chemical analysis in order to identify and characterize drug-lipid interactions that may profoundly affect liposome properties and performance early in the formulation development process.