Headgroup specificity for the interaction of the antimicrobial peptide tritrpticin with phospholipid Langmuir monolayers

We examined the interaction of the cationic antimicrobial peptide (AMP) tritrpticin (VRRFPWWWPFLRR, TRP3) with Langmuir monolayers of zwitterionic (dipalmitoyl phosphatidylcholine, DPPC, and dipalmitoyl phosphatidylethanolamine, DPPE) and negatively charged phospholipids (dipalmitoyl phosphatidic acid, DPPA, and dipalmitoyl phosphatidylglycerol, DPPG). Both surface pressure and surface potential isotherms became more expanded upon addition of TRP3 (DPPE similar to DPPC << DPPA < DPPG). The stronger interaction with negatively charged phospholipids agrees with data for vesicles and planar lipid bilayers, and with AMPs greater activity against bacterial membranes versus mammalian cell membranes. Considerable expansion of negatively charged monolayers occurred at 10 and 30 mol% TRP3, especially at low surface pressure. Moreover, a difference was observed between PA and PG, demonstrating that the interaction, besides being modulated by electrostatic interactions, displays specificity with regard to headgroup, being more pronounced in the case of PG, present in large quantities in bacterial membranes. In previous studies, it was proposed that the peptide acts by a toroidal pore-like mechanism [1,2]. Considering the evidence from the literature that PG shows a propensity to form a positive curvature as do toroidal pores, the observation of TRP3's preference for the PG headgroup and the dramatic increase in area promoted by this interaction represent further support for the toroidal pore mechanism of action proposed for TRP3. (C) 2012 Elsevier B.V. All rights reserved.

Interaction of chitosan and mucin in a biomembrane model environment

Chitosans have been widely exploited in biological applications, including drug delivery and tissue engineering, especially owing to their mucoadhesive properties, but the molecular-level mechanisms for the chitosan action are not known in detail. It is believed that chitosan could affect the mucus by interacting with the proteins mucins, in a process mediated by the cell membrane. In this study we used Langmuir monolayers of dimyristoylphosphatidic acid (DMPA) as simplified membrane models to investigate the interplay between the activity of mucins and chitosan. Surface pressure and surface potential measurements were performed with DMPA monolayers onto which chitosan and/or mucin was adsorbed. We found that the expanding effect from mucin was considerably reduced when chitosan was injected after mucin had been adsorbed on the DMPA monolayer. The results were consistent with the formation of complexes between mucin and chitosan, thus highlighting the importance of electrostatic interactions. Furthermore, chitosan could remove mucin that was co-deposited along with DMPA in Langmuir-Blodgett (LB) films, which could be ascribed to molecular-level interactions between chitosan and mucin inferred from the FTIR spectra of the LB films. In conclusion, the results with Langmuir and LB films suggest that electrostatic interactions are crucial for the mucoadhesive mechanism, which is affected by the complexation between chitosan and mucin. (C) 2012 Elsevier Inc. All rights reserved.

Influence of the type of phospholipid head and of the conformation of the polyelectrolyte on the growth of calcium carbonate thin films on LB/LbL matrices

Calcium carbonate is one of the most important biominerals, and it is the main constituent of pearls, seashells, and teeth. The in vitro crystallization of calcium carbonate using different organic matrices as templates has been reported. In this work, the growth of calcium carbonate thin films on special organic matrices consisting of layer-by-layer (LbL) polyelectrolyte films deposited on a pre-formed phospholipid Langmuir-Blodgett (LB) film has been studied. Two types of randomly coiled polyelectrolytes have been used: lambda-carrageenan and poly(acrylic acid). A precoating comprised of LB films has been prepared by employing a negatively charged phospholipid, the sodium salt of dimyristoilphosphatidyl acid (DMPA), or a zwitterionic phospholipid, namely dimyristoilphosphatidylethanolamine (DMPE). This approach resulted in the formation of particulate calcium carbonate continuous films with different morphologies, particle sizes, and roughness, as revealed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The crystalline structure of the calcium carbonate particles was analyzed by Raman spectroscopy. The randomly coiled conformation of the polyelectrolytes seems to be the main reason for the formation of continuous films rather than CaCO3 isolated crystals. (C) 2012 Elsevier B.V. All rights reserved.

Effect of cations/polycations on the efficiency of formation of a hybrid bilayer membrane that mimics the inner mitochondrial membrane

We aim in this study to characterize the effect of cations and polycations on the formation of hybrid bilayer membranes (HBMs), especially those that mimic the inner mitochondrial membrane (IMM), with a proper composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) adsorbed on an alkanethiol monolayer. HBMs are versatile membrane mimetics that show promising results in sensor technology. Its formation depends on the fusion of vesicles on hydrophobic surfaces, a process that is not well understood at the molecular level. Our results showed to which extend and in which condition the presence of cations and polycations facilitate the formation of HBMs. The required time for lipid layer formation was reduced several times and the lipid layer reaches the expected thickness of 19.5 +/- 1.8 angstrom, in contrast to only 2 +/- 1.5 angstrom usually observed in the absence of cations. In the presence of specific concentrations of spermine and Ca2+ the amount of adsorbed phospholipids on the thiol layer increased nearly 70% compared to that observed when Na+ was used at concentrations 10 times higher. Divalent cations and polycations adsorb specifically on the lipid headgroups destabilizing the hydration forces, facilitating the process of vesicle fusion and formation of lipid monolayers. The concepts and conditions described in the manuscript will certainly help the development of the field of membrane biosensors. (C) 2011 Elsevier B.V. All rights reserved.

The role of cytoskeleton, components of inositol phospholipid signaling pathway and iron in Ehrlichia canis in vitro proliferation

Ehrlichia canis, etiologic agent of Canine Monocytic Ehrlichiosis, is an obligatory intracellular bacterium that parasitizes monocytes and macrophages. In this study we analyzed the role of the cytoskeleton specifically actin microfilaments and microtubules, components of inositol phospholipid signaling pathway such as phospholipase C (PLC), protein kinase (PTK) and calcium channels as well as the role of iron in the E. canis proliferation in DH82 cells. Different inhibitory compounds were used for each component: Cytochalasin D (inhibits actin polymerization), Nocodazole (inhibits microtubule polymerization), Neomycin (PLC inhibitor), Genistein (PTK inhibitor), Verapamil (calcium channel blocker) and Deferoxamine (iron chelator). We observed a significant decrease in the total number of bacteria in infected cells treated suggesting that these cellular components analized are essentials to E. canis proliferation.

Plasma lipases and lipid transfer proteins increase phospholipid but not free cholesterol transfer from lipid emulsion to high density lipoproteins.

Abstract Background Plasma lipases and lipid transfer proteins are involved in the generation and speciation of high density lipoproteins. In this study we have examined the influence of plasma lipases and lipid transfer protein activities on the transfer of free cholesterol (FC) and phospholipids (PL) from lipid emulsion to human, rat and mouse lipoproteins. The effect of the lipases was verified by incubation of labeled (3H-FC,14C-PL) triglyceride rich emulsion with human plasma (control, post-heparin and post-heparin plus lipase inhibitor), rat plasma (control and post-heparin) and by the injection of the labeled lipid emulsion into control and heparinized functionally hepatectomized rats. Results In vitro, the lipase enriched plasma stimulated significantly the transfer of 14C-PL from emulsion to high density lipoprotein (p<0.001) but did not modify the transfer of 3H-FC. In hepatectomized rats, heparin stimulation of intravascular lipolysis increased the plasma removal of 14C-PL and the amount of 14C-PL found in the low density lipoprotein density fraction but not in the high density lipoprotein density fraction. The in vitro and in vivo experiments showed that free cholesterol and phospholipids were transferred from lipid emulsion to plasma lipoproteins independently from each other. The incubation of human plasma, control and control plus monoclonal antibody anti-cholesteryl ester transfer protein (CETP), with 14C-PL emulsion showed that CETP increases 14C-PL transfer to human HDL, since its partial inhibition by the anti-CETP antibody reduced significantly the 14C-PL transfer (p<0.05). However, comparing the nontransgenic (no CETP activity) with the CETP transgenic mouse plasma, no effect of CETP on the 14C-PL distribution in mice lipoproteins was observed. Conclusions It is concluded that: 1-intravascular lipases stimulate phospholipid transfer protein mediated phospholipid transfer, but not free cholesterol, from triglyceride rich particles to human high density lipoproteins and rat low density lipoproteins and high density lipoproteins; 2-free cholesterol and phospholipids are transferred from triglyceride rich particles to plasma lipoproteins by distinct mechanisms, and 3 - CETP also contributes to phospholipid transfer activity in human plasma but not in transgenic mice plasma, a species which has high levels of the specific phospholipid transfer protein activity.

Theoretical Study of the Molecules in Solution and Phospholipid Bilayers

Using Molecular Dynamics simulations and Quantum Mechanical calculations, we study the behavior of molecules with biophysical and pharmacological interest in solution and in phospholipid bilayer.

Langmuir films containing ibuprofen and phospholipids

This study shows the incorporation of ibuprofen, an anti-inflammatory drug, in Langmuir monolayers as cell membrane models. Significant effects were observed for dipalmitoyl phosphatidyl choline (DPPC) monolayers with relevant changes in the elasticity of the monolayer. Dipalmitoyl phosphatidyl glycerol (DPPG) monolayers were affected by small concentrations of ibuprofen, from 1 to 5 mol%. For both types of monolayer, ibuprofen could penetrate into the hydrophobic part of the monolayer, which was confirmed with polarization-modulated infrared reflection–absorption spectroscopy (PM-IRRAS). Brewster angle microscopy (BAM) images showed that ibuprofen prevents the formation of large domains of DPPC. The pharmacological action should occur primarily with penetration of ibuprofen via electrically neutral phospholipid headgroups of the membrane.

Herstellung und physikochemische Charakterisierung von planaren gestützten Lipid-Modellmembran-Systemen

Es wurden funktionalisierte polymerunterstützte planare Phospholipid-Modellmembran-Systeme hergestellt und auf jeder Präparationsstufe eingehend charakterisiert. Dünne Polysaccharidfilme wurden in der Form von quellbaren Gelen auf oxidische Oberflächen aufgebracht und bezüglich ihres Quellungsverhaltens und der Oberflächeneigenschaften in Abhängigkeit vom Wassergehalt untersucht. Lipidmonoschichten unterschiedlicher Zusammensetzung wurden mittels Langmuir-Blodgett-Tranfer auf Polymersubstrate übertragen und bezüglich der Stärke der Lipid/Polymer Wechselwirkung, der lateralen Selbstdiffusion in Abhängigkeit von der Wasseraktivität, dem Spreitverhalten der monomolekularen Membran auf dem Substrat in Abhängigkeit von der Wasseraktivität und dem Lateraldruck der Monoschicht, sowie des Ausmaßes der Hydratation im Kopfgruppenbereich der Lipidmembran in Abhängigkeit von der Wasseraktivität mittels Fluoreszensondenmethoden (Fluoreszenzerholung nach Photobleichung (FRAP), Fluoreszenzmikroskopie und Fluoreszenzspektroskopie) untersucht. Diffusions- und Spreitverhalten von amphiphilen Monoschichten auf Polymersubstraten wurden auf der Basis von in dieser Arbeit entwickelten physikalischen Modellen diskutiert. Mittels Langmuir-Schäfer Transfer wurde auf polymerunterstützte Lipidmonoschichten eine zweite Monoschicht übertragen. Die somit erhaltenen Lipid-Doppelschichtmembranen wurden bezüglich ihrer Stabilität, der lateralen Struktur, der lateralen Selbstdiffusion, des Spreitverhaltens auf unbedeckte Bereiche sowie der Stärke der Membran/Substrat Wechselwirkung vermittels Fluoreszenzmikroskopie, FRAP und Interferenz-Kontrast-Mikroskopie (RICM) untersucht. Schließlich wurden substratgestützte Doppelschicht-Lipidmembranen mit als Protonenpumpen fungierenden integralen Membranproteinen versehen. Die laterale Selbstdiffusion der rekonstituierten Proteinmoleküle wurde mittels FRAP, die funktionale Aktivität der Protonenpumpen mit einem Ionen-sensitiven Feldeffekttransistor-Array analysiert.

Serumfreie Collagen-Monolayer- und Sandwich-Kulturen primärer Hepatozyten als ein wertvolles Modell zur Detektion von Toxizität und Arzneimittelwechselwirkungen in vitro

In dieser Arbeit wurden Zellkulturen primärer Hepatozyten von Ratte und Mensch hinsichtlich ihrer Eignung untersucht Speziesunterschiede der toxischen Wirkung und des Metabolismus von Substanzen darzustellen und inwieweit die in vitro-Ergebnisse in vivo vergleichbar bzw. übertragbar sind. Des Weiteren wurde ein Zellkulturmodell entwickelt, das eine Kultivierung von primären Hepatozyten aus Ratte, Mensch und Maus über einen Zeitraum von mindestens einer bis zwei Wochen erlaubt.rnrnDie Zellkulturen primärer Hepatozyten von Ratte und Mensch zeigten deutliche Unterschiede in der substanzinduzierten Veränderung der Genexpression nach Behandlung mit den, vor allem für den Menschen, lebertoxischen Substanzen Diclofenac und Troglitazon. Diese Unterschiede traten hauptsächlich in der Induktion fremdstoffmetabolisierender Enzyme sowie deren transkriptionsregulierenden Kernrezeptoren in den humanen Hepatozyten auf. Ebenso war eine verstärkte Stressantwort zu beobachten.rnDeutliche Speziesunterschiede konnten ebenso in der Wirkung der Arzneimittelentwicklungssubstanz EMD 392949 auf die Aktivität bzw. Genexpression von Cytochrom P450 Enzymen sowie deren Regulatoren nachgewiesen werden. Des Weiteren konnte hier eine sehr gute Übereinstimmung der Ergebnisse aus den Zellkulturen primärer Ratten- bzw. Humanhepatozyten mit jenen aus in vivo-Experimenten mit Ratten bzw. Affen (Macaca fascicularis) beobachtet werden, was die Aussagekraft der Primärkulturen verdeutlichte.rnDie große Übereinstimmung zwischen Enzymaktivität und Genexpression in der Induktion fremdstoffmetabolisierender Enzyme konnte durch die Behandlung mit einer Reihe speziesspezifischer Induktoren in Zellkulturen primärer Ratten- bzw. Humanhepatozyten bestätigt werden; vor allem nach dem von der amerikanischen Arzneimittelzulassungsbehörde (FDA, Food and Drug Administartion) vorgeschlagenen Bewertungsschema zur Untersuchung der CYP-Induktion.rnrnDie Lebensdauer sowie der Differenzierungsgrad von primären Hepatozyten in Kultur sind stark abhängig von den Zellkulturbedingungen. Durch diese Arbeit konnte gezeigt werden, dass spezifische Eigenschaften von Rattenleberzellen durch Kultivierung in einem Sandwich aus zwei hydratisierten Collagengelschichten und unter serumfreien Bedingungen für einen Zeitraum von mindestens zwei Wochen aufrechterhalten werden können. Dieses Kulturmodel konnte auf Primärhepatozyten von Mensch und Maus übertragen werden und erweitert die möglichen Anwendungen hin zu einer Behandlung über einen längeren Zeitraum und der Untersuchung von subchronischen Effekten.rn

Trypanosoma brucei: a model micro-organism to study eukaryotic phospholipid biosynthesis

Although the protozoan parasite, Trypanosoma brucei, can acquire lipids from its environment, recent reports have shown that it is also capable of de novo synthesis of all major phospholipids. Here we provide an overview of the biosynthetic pathways involved in phospholipid formation in T. brucei and highlight differences to corresponding pathways in other eukaryotes, with the aim of promoting trypanosomes as an attractive model organism to study lipid biosynthesis. We show that de novo synthesis of phosphatidylethanolamine involving CDP-activated intermediates is essential in T. brucei and that a reduction in its cellular content affects mitochondrial morphology and ultrastructure. In addition, we highlight that reduced levels of phosphatidylcholine inhibit nuclear division, suggesting a role for phosphatidylcholine formation in the control of cell division. Furthermore, we discuss possible routes leading to phosphatidylserine and cardiolipin formation in T. brucei and review the biosynthesis of phosphatidylinositol, which seems to take place in two separate compartments. Finally, we emphasize that T. brucei represents the only eukaryote so far that synthesizes all three sphingophospholipid classes, sphingomyelin, inositolphosphorylceramide and ethanolaminephosphorylceramide, and that their production is developmentally regulated.

Ginger phenylpropanoids inhibit IL-1beta and prostanoid secretion and disrupt arachidonate-phospholipid remodeling by targeting phospholipases A2

The rhizome of ginger (Zingiber officinale) is employed in Asian traditional medicine to treat mild forms of rheumatoid arthritis and fever. We have profiled ginger constituents for robust effects on proinflammatory signaling and cytokine expression in a validated assay using human whole blood. Independent of the stimulus used (LPS, PMA, anti-CD28 Ab, anti-CD3 Ab, and thapsigargin), ginger constituents potently and specifically inhibited IL-1β expression in monocytes/macrophages. Both the calcium-independent phospholipase A(2) (iPLA(2))-triggered maturation and the cytosolic phospholipase A(2) (cPLA(2))-dependent secretion of IL-1β from isolated human monocytes were inhibited. In a fluorescence-coupled PLA(2) assay, most major ginger phenylpropanoids directly inhibited i/cPLA(2) from U937 macrophages, but not hog pancreas secretory phospholipase A(2). The effects of the ginger constituents were additive and the potency comparable to the mechanism-based inhibitor bromoenol lactone for iPLA(2) and methyl arachidonyl fluorophosphonate for cPLA(2), with 10-gingerol/-shogaol being most effective. Furthermore, a ginger extract (2 μg/ml) and 10-shogaol (2 μM) potently inhibited the release of PGE(2) and thromboxane B2 (>50%) and partially also leukotriene B(4) in LPS-stimulated macrophages. Intriguingly, the total cellular arachidonic acid was increased 2- to 3-fold in U937 cells under all experimental conditions. Our data show that the concurrent inhibition of iPLA(2) and prostanoid production causes an accumulation of free intracellular arachidonic acid by disrupting the phospholipid deacylation-reacylation cycle. The inhibition of i/cPLA(2), the resulting attenuation of IL-1β secretion, and the simultaneous inhibition of prostanoid production by common ginger phenylpropanoids uncover a new anti-inflammatory molecular mechanism of dietary ginger that may be exploited therapeutically.

myo-Inositol uptake is essential for bulk inositol phospholipid but not glycosylphosphatidylinositol synthesis in Trypanosoma brucei

myo-Inositol is an essential precursor for the production of inositol phosphates and inositol phospholipids in all eukaryotes. Intracellular myo-inositol is generated by de novo synthesis from glucose 6-phosphate or is provided from the environment via myo-inositol symporters. We show that in Trypanosoma brucei, the causative pathogen of human African sleeping sickness and nagana in domestic animals, myo-inositol is taken up via a specific proton-coupled electrogenic symport and that this transport is essential for parasite survival in culture. Down-regulation of the myo-inositol transporter using RNA interference inhibited uptake of myo-inositol and blocked the synthesis of the myo-inositol-containing phospholipids, phosphatidylinositol and inositol phosphorylceramide; in contrast, it had no effect on glycosylphosphatidylinositol production. This together with the unexpected localization of the myo-inositol transporter in both the plasma membrane and the Golgi demonstrate that metabolism of endogenous and exogenous myo-inositol in T. brucei is strictly segregated.

Increased placental phospholipid levels in pre-eclamptic pregnancies

Physiological pregnancy is associated with an increase in lipids from the first to the third trimester. This is a highly regulated response to satisfy energy and membrane demands of the developing fetus. Pregnancy disorders, such as pre-eclampsia, are associated with a dysregulation of lipid metabolism manifesting in increased maternal plasma lipid levels. In fetal placental tissue, only scarce information on the lipid profile is available, and data for gestational diseases are lacking. In the present study, we investigated the placental lipid content in control versus pre-eclamptic samples, with the focus on tissue phospholipid levels and composition. We found an increase in total phospholipid content as well as changes in individual phospholipid classes in pre-eclamptic placental tissues compared to controls. These alterations could be a source of placental pathological changes in pre-eclampsia, such as lipid peroxide insult or dysregulation of lipid transport across the syncytiotrophoblast.