Recently, three distinct isoforms of phospholipase D (PLD) were identified in Arabidopsis thaliana. PLD α represents the well-known form found in plants, while PLD β and γ have been only recently discovered (Pappan et al., 1997b; Qin et al., 1997). These isoforms differ in substrate selectivity and cofactors required for activity. Here, I report that PLD β and γ isoforms were active toward N-acylphosphatidylethanolamine (NAPE), but PLD α was not. The ability of PLD β and γ to hydrolyze NAPE marks a key difference from PLD α. N-acylethanolamines (NAE), the hydrolytic products of NAPE by PLD β and γ, inhibited PLD α from castor bean and cabbage. Inhibition of PLD α by NAE was dose-dependent and inversely proportional to acyl chain length and degree of unsaturation. Enzyme kinetic analysis suggested non-competitive inhibition of PLD α by NAE 14:0. In addition, a 1.2-kb tobacco (Nicotiana tabacum L.) cDNA fragment was isolated that possessed a 74% amino acid identity to Arabidopsis PLD β indicating that this isoform is expressed in tobacco cells. Collectively, these results provide evidence for NAE producing PLD activities and suggest a possible regulatory role for NAE with respect to PLD α.

Although phosphatidylinositol (PI) and phosphoinositides (PIPs) only comprise a small percentage of the inner leaflet of the plasma membrane, they mediate a large variety of signaling events. In previous studies, we have observed the absence of macroscopically discernible domains in mixtures of PI/PC and PI(4,5)P2/PC. The addition of cholesterol to these mixtures results in condensation of the monolayer and hence domain formation. To better mimic the ionic conditions and hydrogen bonding properties of the inner leaflet plasma membrane, we investigated in this study the effect of common inner leaflet plasma membrane lipids like phosphatidylethanolamine (PE), phosphatidylserine (PS) and PI, on phosphoinositide domain behavior in the presence of cholesterol and/or bivalent cations.

The goal of this doctoral work was to observe and analyze the stability and dynamics of phospholipid-encapsulated microbubbles, and in particular the reaction to sudden submersion in a multi-gas medium. To accomplish this goal, first an experimental technique was developed to observe a microbubble in a single-gas environment suddenly immersed in a multi-gas environment, without perturbing the microbubble position. A modified Epstein-Plesset model was concurrently developed to account for the multiple gas species in the bulk solution. The model was used to analyze previous data for the effect of anesthesia carrier gas on microbubble ultrasound contrast agent in vivo circulation persistence. The focus of the experiments then shifted to microbubbles of different sizes encapsulated with a homologous series of saturated diacyl-chain lipid surfactants and emulsifiers. Constitutive models for the elastic and gas permeation properties of the lipid encapsulation were developed to elucidate the unique behaviors observed during the experiments. The experimental techniques employed were: (1) transmission bright field optical microscopy to obtain real-time, digital videos of microbubbles growing and dissolving in response to perturbations in the local gas environment and (2) the Langmuir trough film balance to determine the elasticity of the phospholipid monolayers during compression, expansion, and expansive relaxation. The modeling techniques employed was (1) a forward-wind finite difference method to discretize a series of non-linear differential equations and (2) a Newton-Raphson method to solve the diameter of a microbubble from the mechanical stress balance. These modeling techniques were used to determine the behavior of a microbubble a priori, whereas the fitting models implemented the iterative methods to solve for parameters without a Newton-Raphson method. Results showed that microbubbles coated with soluble surfactants and dissolving in a single gas solution could be predicted by the original Epstein-Plesset model. When subjected to a multi-gas medium, the modified Epstein-Plesset model accurately predicted microbubble growth and dissolution. The model was used to analyze the increase in microbubble circulation lifetime observed by others in anesthetized rats inhaling air rather than oxygen as the anesthesia carrier gas. The predictive capabilities of the model broke down, however, if the gas-core was encapsulated with a phospholipid monolayer. A typical, large (>40 µm diameter) lipid-coated microbubble displayed stunted growth, followed by three anomalous dissolution regimes: (1) rapid dissolution back to the initial resting diameter followed by (2) slow, steady dissolution and finally (3) stabilization, where the apparent surface tension approached a near-zero value. The model was modified to allow fitting of the radius-time curve by varying the surface tension. The analysis showed that the surface tension is dynamic, and suggested that a "break up" tension allowed for rapid expansion of the microbubble beyond the initial resting diameter. Lipid jamming was proposed as the mechanism eventually halting dissolution. Further observations of smaller microbubbles (<20 µm diameter) coated with a homologous series of saturated diacyl chain lipids gave significantly different results. Initially the microbubbles grew, but growth was severely subdued, if not eliminated, for more solid encapsulations below a threshold size (~10 µm diameter). Following growth, most microbubbles rapidly dissolved back to their original size. The microbubbles then experienced an anomalous lag time before spontaneously dissolving again. The lag times were highly variable and shown to correlate to the reduced temperature of the encapsulation, rather than the initial microbubble size. Most of the microbubbles stabilized again at a diameter of 1-2 µm, and this "stable diameter" appeared to be universal and independent of both the initial microbubble size and the rigidness of the encapsulation. Constitutive models were developed to describe these physical phenomena in the early growth and dissolution stages which were verified with independent monolayer relaxation studies.

Abstract not available

Severe dehydration is lethal for most biological species, however there are a number of organisms which have evolved mechanisms to avoid damage during dehydration. One of these mechanisms is the accumulation of small solutes (e.g. sugars), which have been shown to preserve membranes by inhibiting deleterious phase changes at low hydration. Specifically, sugars reduce the gel to fluid phase transition temperatures of model lipid/water mixtures. However, there is debate about the precise mechanism, the resolution of which hinges on the location of the sugars. An experimental investigation into the effects of small solutes on the phase behaviour of phospholipid membranes is presented in order help identify the mechanisms by which solutes facilitate desiccation tolerance. Differential Scanning Calorimetry (DSC) was used to determine the first comprehensive phase diagram for the synthetic phospholipid DPPC over a wide range of hydration and solute molar ratios between 0.1 and 1.0 sugars per lipid. Over the same range of hydrations and solute concentrations Small Angle X-Ray Scattering (SAXS) was used to measure the structural parameters of the membrane bilayers necessary to determine both the phase of the membrane lipids and the location of the solutes. SAXS was also used to conduct the first comprehensive study of the effect of solutes on the kinetics of the fluid - gel transition of DPPC over a range of both hydration and solute concentration. Finally, contrast variation Small Angle Neutron Scattering (SANS) was used to quantitatively determine the location of the solutes. Data from these complimentary techniques are presented which show a monotonic relationship between both transition temperature and repeat spacing with respect to solute concentration. This relationship exists between solute:lipid molar ratios between 0.1 to approximately 0.5, after which higher concentrations of solute are shown to have no further effect on either the bilayer repeat spacing or transition temperature. It is proposed that the exclusion of small solutes into sugar/water micro-phases external to the bilayer can account for this behaviour. A theoretical model previously used to describe membrane phase behaviour at low hydrations is modified to account for the presence of solutes between membrane bilayers. This model is shown to be in quantitative agreement with the experimental data up until approximately 0.5 sucrose molecules per lipid, the point of solute exclusion. Once exclusion is taken into account, the model is quantitative over the whole range of sugar concentrations.

Anthocyanins are the most important class of water-soluble pigments responsible for red to blue colors in various plants. Anthocyanins naturally occur in a broad range of plants and studies have shown associations between fruit consumption and reduction of certain diseases thought to be related to the presence of these and other polyphenolics. However, anthocyanin absorption is fairly poor which hinders their potential to be utilized in the human body. Absorption of anthocyanins extracted from açaí puree and port wine was assessed. Various combinations of terpenes and phospholipids were added to anthocyanins to modulate and increase their transport within a model system. Açaí and port wine anthocyanins were poorly transported in the absence of phospholipids and terpenes. The addition of terpenes and phospholipids significantly increased the transport of anthocyanins. Additionally, the presence of phospholipids and terpenes did not influence the way anthocyanins degraded over a 40 day period of time at three different temperatures. Transport of anthocyanins was not dependent on dosage since absorption results were similar at both concentrations of anthocyanins tested. Two methods to mix anthocyanins, phospholipids, and terpenes were also assessed (Sonication and French Press). Comparisons illustrated that both technologies created matrices that maintained the properties of phospholipids and terpenes as transport enhancers. Finally, a study to determine the efficacy of phospholipids and terpenes on a different type of polyphenolic compound was assessed. Transport of gallic acid was enhanced by the use of these agents that cemented the idea that phospholipids and terpenes can enhance the transport of various types of polyphenolics. The aiding effect of phospholipids and terpenes was well established and could play an important role in future investigation in this field. Further research needs to be conducted to reveal more information about the nature of these vesicles or associations that phospholipids and terpenes may have with anthocyanins. In vivo studies need to be considered to confirm these effects in rat models and, ideally, in humans. Nevertheless, these findings open a new line of investigation that could harvest promising results for the future of ingredient development for food products.

Structured phospholipids with functional ingredients like conjugated linoleic acid (CLA) and plant sterols to deliver their physiological effects in different food formulations were synthesized. The lipase and phospholipase A2 catalyzed enzymatic acidolysis reaction between phospholipids (PLs) and CLA was used for fatty acid modification, while the phospholipase D catalyzed transphosphatidylation reaction between PLs and sterol was used for head group modification. Enzymatic processes were an effective way to produce structured phospholipids. Screening of four lipases and immobilized phospholipase A2 and combination of lipase and phospholipase showed that only Lipozyme RM IM and Lipozyme TL IM were effective in incorporation of CLA into PLs. The maximum incorporation achieved by the latter enzyme was 16% with soy PLs in 72 h. The class of phospholipids had a significant effect on the rate of incorporation of CLA compare to source of PLs. A method capable of predicting the rate of incorporation of CLA into phospholipids was developed using response surface methodology. A three-level four-factor Central Composite Rotatable Design (CCRD) was used. The four factors selected were lipase dosage (Ed, wt.% of substrate), substrate ratio (Sr,mol%), reaction time (ti, h) and reaction temperature (Te,oC). The enzyme load and substrate ratio had a greater effect on the rate of incorporation than did reaction time and temperature. A polynomial regression equation was developed to predict the reaction rate. The new phosphatidyl derivative, phosphatidyl-sitosterol, was found to be synthesized by the transfer reaction of phosphatidyl residue from phosphatidylcholine to &#946;-sitosterol by phospholipase D from Streptomyces sp. in biphasic medium. The novel phosphatidyl .sitosterol derivative was identified by MALDI-TOF mass spectrometry. Plant sterols were modified to a more polar lipid class by synthesizing phospholipid derivatives of them. When these structured phospholipids were added to a whey protein based oil-in-water emulsion, the CLA incorporated structured phospholipids (CLA-PL) had higher heat stability and oxidative stability compared to the controls.

Thesis (M.S.)--University of North Carolina at Wilmington, 2003.

No description available.

Fettleibigkeit und Adipositas haben in den letzten Jahren weltweit drastisch zugenommen. Die Fettleibigkeit geht nicht nur mit einer verringerten Lebensqualität einher, sondern ist auch mit verschiedenen Folgeerkrankungen, wie kardiovaskulären Erkrankungen (z.B. Arteriosklerose) und metabolische Erkrankungen (z.B. Diabetes) assoziiert. Vorliegende Studien belegen einen Zusammenhang zwischen Diabetes und männlicher Infertilität. In der vorliegenden Arbeit wurden daher die Spermienmembran wie auch das Seminalplasma (SP) mittels matrix-assisted laser desorption and ionisation time-of-flight Massenspektrometrie (MALDI-TOF MS) analysiert, um mögliche Lipid-Biomarker für die Fertilität bzw. Infertilität zu finden. Dafür wurde zunächst die MALDI-TOF MS Methode mit relevanten Standardlipiden optimiert. Anschließend konnte sowohl das Phospholipidmuster des SP mit dem Spermien verglichen werden als auch die Spermienlipide von normalgewichtigen und fettleibigen Probanden. Durch diese Analyse konnte das Phosphatidylcholin/Lysophosphatidylcholin (PC/LPC)-Verhältnis, aber auch ein stark erhöhter Sphingomyelin (SM)-Anteil bei den fettleibigen Probanden als Qualitätsmarker gefunden werden. Des Weiteren wurden im Rahmen dieser Arbeit murine Spermien aus dem Caput und dem Cauda der Epididymis mittels MALDI-TOF MS analysiert und das Phospholipidmuster miteinander verglichen. Es konnte damit gezeigt werden, dass die murinen Spermien einen wesentlich höheren Anteil an Stearinsäureresten aufweisen, die humanen Spermien hingegen vor allem durch Palmitinsäurereste charakterisiert sind. In den Spermienmembranen aus dem Cauda und dem Caput gab es wesentliche Unterschiede im Phospholipidmuster. Spermienmembranen aus dem Caput besitzen einen hohen Anteil an PC und Phosphatidylethanolamin (PE). Die Spermienmembranen aus dem Cauda hingegen enthalten mehr SM, und auch einen höheren Anteil an LPCs und Formyl-LPC. Diese Arbeit konnte somit zeigen, dass die Reifungsprozesse in der Epididymis auch die Phospholipid-Zusammensetzung betreffen.