Facile Lipid Flip-Flop in a Phospholipid Bilayer Induced by Gramicidin A Measured by Sum-Frequency Vibrational Spectroscopy

The first direct experimental evidence that gramicidin A (gA), a transmembrane peptide, facilitates the translocation of unlabeled lipids in a phospholipid bilayer was obtained with sum-frequency vibrational spectroscopy (SFVS). SFVS was used to investigate the effect of gA on lipid flip-flop in a planar 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipid bilayer. The kinetics of lipid translocation were determined by an analysis of the SFVS intensity versus time at different temperatures in the presence of 2 mol % gA. The rate constants of DSPC flip-flop increase from 2 to 10 times relative to the pure DSPC system. The results indicate that facial lipid exchange can be induced by a hydrophobic transmembrane helix. The increase in lipid flip-flop rates is correlated to an increase in the gauche content of the lipid tails. The results suggest that membrane defects induced by the presence of integral membrane proteins may play a large role in modulating the rate of lipid flip-flop.     

Plant Water Potential Gradients Measured in the Field by Freezing Point

A portable freezing point meter was used in the field to measure the water potential gradients in sunflower (Helianthus annuus), beans (Phaseolus vulgaris), corn (Zea mays), wheat (Triticum aestivum), pumpkin (Cucurbita pepo), potato (Solanum tuberosum), alfalfa (Medicago sativa), and sugarbeets (Beta vulgaris). The measurements were made between daybreak and sunrise, and again during the middle of the afternoon on days when the potential evapotranspiration varied between 6.5 and 8.0 mm of water. The gradients varied from a maximum of 0.2 bar per cm in a wheat, down to an undetectable value for pumpkin. Although most of the soil in the root zone was kept at potentials above –1 bar, the bulk of the root tissue had water potentials of –5 to –10 bars. Differences in water potential between shaded and unshaded leaves, and between leaf tissue and guttation fluid suggested a similar drop of several bars between xylem elements and the surrounding leaf tissue in some plant species. The implications of such drops are discussed with respect to plant water transport equations and pressure cell potential measurements.     

Effect of Long-Term Feeding with Acetyl-L-Carnitine on the Age-Related Changes in Rat Brain Lipid Composition: A Study by 31P NMR Spectroscopy

Changes in brain lipid composition have been determined in 24 months-old Fischer rats with respect to 6 months-old ones. The cerebral levels of sphingomyelin and cholesterol were found to be significantly increased in aged rats, whereas the amount of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid appear to be unaffected by aging. Long-term feeding with acetyl-L-carnitine was able to reduce the age-dependent increase of both sphingomyelin and cholesterol cerebral levels with no effect on the other measured phospholipids. These findings shown that changes in membrane lipid metabolism and/or composition represent one of the alterations occurring in rat brain with aging, and that long-term feeding with acetyl-L-carnitine can be useful in normalizing these age-dependent disturbances.     

Molecular Adsorption Steers Bacterial Swimming at the Air/Water Interface

Microbes inhabiting Earth have adapted to diverse environments of water, air, soil, and often at the interfaces of multiple media. In this study, we focus on the behavior of Caulobacter crescentus, a singly flagellated bacterium, at the air/water interface. Forward swimming C. crescentus swarmer cells tend to get physically trapped at the surface when swimming in nutrient-rich growth medium but not in minimal salt motility medium. Trapped cells move in tight, clockwise circles when viewed from the air with slightly reduced speed. Trace amounts of Triton X100, a nonionic surfactant, release the trapped cells from these circular trajectories. We show, by tracing the motion of positively charged colloidal beads near the interface that organic molecules in the growth medium adsorb at the interface, creating a high viscosity film. Consequently, the air/water interface no longer acts as a free surface and forward swimming cells become hydrodynamically trapped. Added surfactants efficiently partition to the surface, replacing the viscous layer of molecules and reestablishing free surface behavior. These findings help explain recent similar studies on Escherichia coli, showing trajectories of variable handedness depending on media chemistry. The consistent behavior of these two distinct microbial species provides insights on how microbes have evolved to cope with challenging interfacial environments.     

Structure and Dynamics of the Myristoyl Lipid Modification of Src Peptides Determined by H Solid-State NMR Spectroscopy

Lipid modifications of proteins are widespread in nature and play an important role in numerous biological processes. The nonreceptor tyrosine kinase Src is equipped with an N-terminal myristoyl chain and a cluster of basic amino acids for the stable membrane association of the protein. We used ²H NMR spectroscopy to investigate the structure and dynamics of the myristoyl chain of myr-Src(2-19), and compare them with the hydrocarbon chains of the surrounding phospholipids in bilayers of varying surface potentials and chain lengths. The myristoyl chain of Src was well inserted in all bilayers investigated. In zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes, the myristoyl chain of Src was significantly longer and appears “stiffer” than the phospholipid chains. This can be explained by an equilibrium between the attraction attributable to the insertion of the myristoyl chain and the Born repulsion. In a 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine] membrane, where attractive electrostatic interactions come into play, the differences between the peptide and the phospholipid chain lengths were attenuated, and the molecular dynamics of all lipid chains were similar. In a much thicker 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine]/cholesterol membrane, the length of the myristoyl chain of Src was elongated nearly to its maximum, and the order parameters of the Src chain were comparable to those of the surrounding membrane.     

Formation of Oil/Water Emulsions due to Electrochemical Instability at the Liquid/Liquid Interface

This study is dedicated to the development of manufacturing principles of food emulsions for food functionalization and formulation. In the experiments presented, the emulsions are generated using the effects of electrochemical instability and surface tension decrease at a polarized liquid/liquid interface. Polarization of the oil/liquid interface is studied in various aqueous systems including electrolytes and water-based solutions of cationic, anionic, zwitterionic, and nonionic surfactants. The physical features of the obtained emulsions, such as hydrodynamic flow patterns, droplet size distribution, and emulsion organization, are described. The efficacy of different electrode configurations is also evaluated. The theoretical model of the liquid/liquid interface polarization and experimental data obtained from various emulsification regimes are presented.

The Equilibria Between Monovalent Ions and Phosphatidylcholine Monolayer at the Air/Water Interface

The effect of monovalent ion (Li⁺, Na⁺, Cs⁺) interaction with monolayers of phosphatidylcholine (lecithin, PC) was investigated at the air/water interface. We present surface tension measurements of lipid monolayers obtained using a Langmuir method as a function of monovalent ion concentration. Measurements were carried out at 22 °C using a Teflon trough and a Nima 9000 tensiometer. Interactions between lecithin and monovalent ions result in significant deviations from the additivity rule. An equilibrium theory to describe the behavior of monolayer components at the air/water interface was developed in order to obtain the stability constants and area occupied by one molecule of PC–monovalent ion complexes (PC^?Me⁺).     

Cerebral Acid Buffering Capacity at Different Ages Measured In Vivo by 31P and 1H Nuclear Magnetic Resonance Spectroscopy

Cerebral acidosis occurring during ischemia has been proposed as one determinant of tissue damage. Newborn animals appear to be less susceptible to ischemic tissue damage than adults. One possible component of ischemic tolerance could derive from maturational differences in the extent of acid production and buffering in newborns compared to adults. The purpose of this study was to measure the dependency of acid production on the blood plasma glucose concentrations and acid buffering capacity of piglets at different stages of development. Complete ischemia was induced in 29 piglets ranging in postconceptual age from 111 to 156 days (normal term conception, 115 days). Brain buffering capacity during the first 30 min of ischemia was quantified in vivo, via 31P and 1H nuclear magnetic resonance (NMR) spectroscopy, by measuring the change in intracellular brain pH for a given change in the concentration of compounds that contribute to the production of hydrogen ions. Animals from all four age groups showed a similar linear correlation between preischemia blood glucose concentration and intracellular pH after 30 min of ischemia. For each animal the slope of the plot of intracellular pH versus cerebral buffer base deficit was used to calculate the buffer capacity. Using data obtained over the entire 30 min of ischemia, there was no difference in the mean buffer capacity of the different age groups, nor was there a significant correlation between buffer capacity and age. However, there was a significant increase in buffer capacity for the intracellular pH range 6.6-6.0, compared to 7.0-6.6, for all age groups. No significant differences in buffer capacity for these two pH ranges were observed between any of the age groups. Acid buffering capacity was also measured by performing pH titrations on brain tissue homogenized in the presence of inhibitors of glycolysis and creatine kinase. Plots of homogenate pH versus buffer base deficit showed a nonlinear trend similar to that seen in vivo, indicating an increase in buffer capacity as intracellular pH decreases. A comparison of newborn and 1-month-old brain tissue frozen under control conditions or after 45 min of ischemia revealed no differences that could be attributed to age and a slight decrease in buffer capacity of ischemic brain compared to control brain tissue homogenates. There was no difference between the brain buffering capacity measured in vivo using 31P and 1H NMR and that measured in vitro using brain homogenates.     

An Alpha-Helical Peptide in AOT Micelles Prefers to be Localized at the Water/Headgroup Interface

A model alpha-helical peptide encapsulated in a reverse micelle is used to study the structure and dynamics of proteins under constrained environments that mimic the membrane-water environment in cells. Molecular dynamics simulations of the self assembly of systems composed of a peptide, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), water, and isooctane show that the peptide prefers to be located at the water/AOT headgroups interface. We explore the effect of the AOT headgroup charge and the peptide charge and find that the peptides migrate to the interface in all cases. These results show that the peptides prefer the constrained hydration environment of the AOT headgroups. The driving force for this configuration is the gain in entropy by released water molecules that otherwise would solvate the protein and surfactant headgroups     

Development of a CP P NMR Broadline Simulation Methodology for Studying the Interactions of Antihypertensive AT1 Antagonist Losartan with Phospholipid Bilayers

A cross-polarization (CP) ³¹P NMR broadline simulation methodology was developed for studying the effects of drugs in phospholipids bilayers. Based on seven-parameter fittings, this methodology provided information concerning the conformational changes and dynamics effects of losartan in the polar region of the dipalmitoylphosphatidylcholine bilayers. The test molecule for this study was losartan, an antihypertensive drug known to exert its effect on AT₁ transmembrane receptors. The results were complemented and compared with those of differential scanning calorimetry, solid-state ¹³C NMR spectroscopy, Raman spectroscopy, and electron spin resonance. More specifically, these physical chemical methodologies indicated that the amphipathic losartan molecule interacts with the hydrophilic-head zone of the lipid bilayers. The CP ³¹P NMR broadline simulations showed that the lipid molecules in the bilayers containing losartan displayed greater collective tilt compared to the tilt displayed by the load-free bilayers, indicating improved packing. The Raman results displayed a decrease in the trans/gauche ratio and increased intermolecular interactions of the acyl chains in the liquid crystalline phase. Additional evidence, suggesting that losartan possibly anchors in the realm of the headgroup, was derived from upfield shift of the average chemical shift σ^iso of the ³¹P signal in the presence of losartan and from shift of the observed peak at 715 cm⁻¹ attributed to C-N stretching in the Raman spectra.     

Structure and Membrane Interactions of the Antibiotic Peptide Dermadistinctin K by Multidimensional Solution and Oriented N and P Solid-State NMR Spectroscopy

DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an α-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with ¹⁵N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting ¹⁵N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled ³¹P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing.     

Competitive Adsorption of Lecithin and Saliva at the O/W Interface in Relation to the Oral Processing of Lipid Continuous Foods

This research is relevant to oral processing of lipid continuous foods. During this first step of food digestion, lipid continuous foods such as chocolate or margarine phase invert into oil-in-water emulsions stimulated through the mechanical action of tongue and teeth in combination with the change in temperature and the high surface activity of salivary proteins. These are hypothesised to stabilise the newly formed interface in competition with surfactants or surface active molecules released from the food if present. Here competitive adsorption between mechanically stimulated human whole saliva (HWS) and lecithin dissolved in sunflower oil freed of interfacially active contaminants was investigated in-vitro using a pendant drop tensiometer for dynamic interfacial tension and interfacial rheological measurements. Initially, it was validated that the interfacial properties of HWS samples remained unaffected by frozen storage at ?80 °C during 6 weeks. Protein concentration affected the absolute values of interfacial tension and in particular the dilatational elastic modulus. Competitive adsorption studies revealed a mixed interface and it follows that emulsion stabilisation during oral processing involves both salivary proteins and lecithin present in the oil phase.     

31P NMR analysis of the DNA conformation induced by protein binding SRY/DNA complexes.

Complexes of the HMG box protein SRY with two duplexes of 8 and 14 base pairs have been studied by 31P NMR and complete assignment of all phosphorus signals of the bound DNA duplexes are presented. While for the free DNA, all 31P signals display limited spectral dispersion (< 0.8 p.p.m.) for the bound duplexes, 31P resonances are spread over 2 p.p.m. Based on the previously published 3D structure of hSRY-HMG, with the 8 mer it is demonstrated that the upfield shifted resonances correspond to the site of partial intercalation of an isoleucine side chain into the DNA. Moreover, the observation of significant difference in linewidths between the two duplexes allows to estimate lifetime of the complexes from 31P-31P 2D exchange experiments.     

Lenstar LS900测量下人工晶状体公式用于高度近视白内障患者的准确性研究

目的:使用Lenstar LS900测量仪对高度近视合并白内障患者进行术前测量,比较SRK/T、Haigis、Barrett Universal II公式的精确性。方法:选择2018年5月至2018年8月于我院行白内障超声乳化吸除及人工晶状体植入术的高度近视合并白内障患者40例(54只眼),根据眼轴的长度分为三组。使用Lenstar LS900对患者进行眼部生物测量,并计算由SRK/T、Haigis、Barrett Universal II公式预测的术后屈光度。将术后一个月得到的实际屈光度与各公式预测的屈光度进行比较分析,观察上述公式在不同眼轴长度组中的准确性。结果:组内各公式的比较:A组:Haigis公式与Barrett Universal II公式间的差异有统计学意义(P0.05),其他公式间比较差异无统计学意义(P0.05);B组:SRK/T公式与Haigis公式比较差异无统计学意义(P0.05),其他公式间比较差异有统计学意义(P0.05);C组:三个公式之间的差异均具有统计学意义(P0.05)。各公式组间比较:SRK/T公式和Haigis公式比较差异具有统计学意义(P0.05),Barrett Universal II公式组间比较差异无统计学意义(P0.05)。结论:Barrett Universal II公式用于高度近视白内障患者在各个眼轴长度的测量中表现出良好的准确性,且准确性不随眼轴的增长而下降。随着眼轴的增长,SRK/T公式的准确性逐渐下降,当AL30 mm时,Haigis公式的准确性优于SRK/T公式。     

Visualization by High Resolution Immunoelectron Microscopy of the Transient Receptor Potential Vanilloid-1 at Inhibitory Synapses of the Mouse Dentate Gyrus

We have recently shown that the transient receptor potential vanilloid type 1 (TRPV1), a non-selective cation channel in the peripheral and central nervous system, is localized at postsynaptic sites of the excitatory perforant path synapses in the hippocampal dentate molecular layer (ML). In the present work, we have studied the distribution of TRPV1 at inhibitory synapses in the ML. With this aim, a preembedding immunogold method for high resolution electron microscopy was applied to mouse hippocampus. About 30% of the inhibitory synapses in the ML are TRPV1 immunopositive, which is mostly localized perisynaptically (∼60% of total immunoparticles) at postsynaptic dendritic membranes receiving symmetric synapses in the inner 1/3 of the layer. This TRPV1 pattern distribution is not observed in the ML of TRPV1 knock-out mice. These findings extend the knowledge of the subcellular localization of TRPV1 to inhibitory synapses of the dentate molecular layer where the channel, in addition to excitatory synapses, is present.     

Interfacial Properties of β-Lactoglobulin at the Oil/Water Interface: Influence of Starch Conversion Products with Varying Dextrose Equivalents

In spray dried emulsions, frequently milk proteins are used as interfacial active components and starch conversion products are added as matrix material at high concentrations. To characterize interfacial properties at the oil/water interface by commonly applied methods, low protein, and carbohydrate concentrations from 1 to 2% are usually analyzed. The impact of a higher concentration of starch conversion products was not investigated so far. Therefore, the formation and rheological properties of β-lactoglobulin (β-LG) stabilized films at the oil/water interface were investigated via short and long-time adsorption behavior using pendant drop tensiometry as well as dilatational and interfacial shear rheology. Suitability of the applied methods to the chosen samples with higher concentrations >1–2% was verified by calculation of selected key numbers like capillary number and by detailed reviewing of the results which is summarized further on as key indicators. It is hypothesized, that the increase in concentration via presence of starch conversion products will delay interfacial stabilization as a result of increased bulk viscosity with decreasing degree of degradation (dextrose equivalent) of the starch. Furthermore, this increase in concentration leads to more stable interfacial films due to thermodynamic incompatibility effects between protein and starch conversion products which results in increases of local protein concentration. Key indicators proved a general suitability of applied methods for the evaluation of the investigated samples. Moreover, results showed an increase in interfacial film stability and elastic properties alongside a decreased interfacial tension if starch conversion products were present in a high concentration.

Graphical abstract

     

Fluorinated End‐Groups in Electrolytes Induce Ordered Electrolyte/Anode Interface Even at Open‐Circuit Potential as Revealed by Sum Frequency Generation Vibrational Spectroscopy

Fluorine‐based additives have a tremendously beneficial effect on the performance of lithium‐ion batteries, yet the origin of this phenomenon is unclear. This paper shows that the formation of a solid‐electrolyte interphase (SEI) on the anode surface in the first five charge/discharge cycles is affected by the stereochemistry of the electrolyte molecules on the anode surface starting at open‐circuit potential (OCP). This study shows an anode‐specific model system, the reduction of 1,2‐diethoxy ethane with lithium bis(trifluoromethane)sulfonimide, as a salt on an amorphous silicon anode, and compares the electrochemical response and SEI formation to its fluorinated version, bis(2,2,2‐trifluoroethoxy) ethane (BTFEOE), by sum frequency generation (SFG) vibrational spectroscopy under reaction conditions. The SFG results suggest that the ? CF₃ end‐groups of the linear ether BTFEOE change their adsorption orientation on the a‐Si surface at OCP, leading to a better protective layer. Supporting evidence from ex situ scanning electron microscopy and X‐ray photoelectron spectroscopy depth profiling measurements shows that the fluorinated ether, BTFEOE, yields a smooth SEI on the a‐Si surface and enables lithium ions to intercalate deeper into the a‐Si bulk.     

Flux control in the rat gastrocnemius glycogen synthesis pathway by in vivo 13C/31P NMR spectroscopy

To determine the relative contributions of glucose transport/hexokinase, glycogen synthase (GSase), and glycolysis to the control of insulin-stimulated muscle glycogen synthesis, we combined 13C and 31P NMR to quantitate the glycogen synthesis rate and glucose 6-phosphate (G-6-P) levels in rat (Sprague-Dawley) gastrocnemius muscle during hyperinsulinemia at euglycemic (E) and hyperglycemic (H) glucose concentrations under thiopental anesthesia. Flux control was calculated using metabolic control analysis. The combined control coefficient of glucose transport/hexokinase (GT/Hk) for glycogen synthesis was 1.1 +/- 0.03 (direct measure) and 1.14-1.16 (calculated for a range of glycolytic fluxes), whereas the control coefficient for GSase was much lower (0.011-0.448). We also observed that the increase in in vivo [G-6-P] from E to H (0.22 +/- 0.03 to 0.40 +/- 0.03 mM) effects a supralinear increase in the in vitro velocity of GSase, from 14.6 to 26.1 mU. kg(-1). min(-1) (1.8-fold). All measurements suggest that the majority of the flux control of muscle glycogen synthesis is at the GT/Hk step.