Lipid-fehérje kölcsönhatás és dinamika vizsgálata modell és biológiai membránokban. Egy új, nanotechnológiás, membránfehérje vizsgáló rendszer kifejlesztése. = Lipid-protein interactions, and dynamics in model and biological membranes. The development of a new experimental nano-technologic device to study structure and functioning of membrane-proteins.

Összehasonlítva a biológiai membránokban a lipid-, illetve a fehérje-dinamika, illetve a fehérjék másodlagos szerkezetváltozásainak hőmérsékletfüggését, megállapítottuk, hogy alacsony hőmérsékleti stressz körülményei között elsősorban a lipidek, magas hőmérsékleti stressz esetén pedig a fehérjék dinamika/szerkezet változásai a meghatározóak a biológiai membrán viselkedésében. (PMC Biophysics (2009) 2:1, Biochemistry (2009) 48, 10120-10128) Polielektrolitokból készített, tetszőlegesen töltött felszíneken felépített egy-molekula vastagságú kazein rétegek adszorpciójának nyomon követésével meghatároztuk a Ca-foszfát szállításáért felelős kazein micellák kialakulásának mechanizmusát. Megmutattuk, hogy a kazein micellák belsejében levő ?-kazein molekulák foszfoszeril csoportjaihoz kapcsolódnak a Ca-foszfát nanoklaszterek, amikhez a további ?-kazeinek pozitív csoportjaikkal kötődnek. Az ?-kazeinek hidrofób csoportjainak kölcsönhatása révén, kis, kb. 20 nm-es aggregátumok alakulnak ki, amelyeket a Ca-foszfát nanoklaszterek kapcsolnak össze. A micellák felszínére ?-kazein molekulák tapadnak, amik “lágyabb“ szerkezetük révén befedik azokat a pontokat az ?-kazeinek felszínén, amik a további akár ?-kazein, akár Ca-foszfát kötődéshez kellenek, és így a micella képződése befejeződik, felszínét ?-kazein molekulák borítják, amik nem érzékenyek a Ca-ionokra, mint az ?-kazein, így a micellák nem csapódnak ki. ( JBC (2010) 285, 38811-38817, Eur Biophys J(2012) 41:959-968) | Using Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy, we have compared lipid and protein dynamics/structural changes as a function of the temperature in biological membranes. It has been shown that among low-temperature stress conditions the changes in the dynamics of the lipids, among high-temperature stress conditions the changes of the protein dynamics and secondary structure are dominant in the behaviour of the membranes. (PMC Biophysics (2009) 2:1, Biochemistry (2009) 48, 10120-10128) By preparing polyelectrolyte films having charged surfaces, and building onto them layer-by-layer aggregated casein-Ca-phosphate complexes, we determined the mechanism of the construction of the casein micelles, the essential components of Ca-phosphate transport in mammals. We have shown that the phosphoseryl residues on the ?-casein molecules are essential for binding the Ca-phosphate. The bound Ca-phosphate nanoclusters bind the next layer of the ?-caseins, as so on. Hydrophobic parts of the amphiphilic molecules organize ?-caseins into about 20 nm diameter aggregates, interconnected by Ca-phosphate nanoclusters. Termination of the micelle formation is achieved via the adsorption of ?-casein onto the micelle surface. Due to its “soft“ secondary structure, ?-casein can cover those parts of the ?-casein molecules, which are essential for Ca-phosphate binding and further aggregation of ?-caseins. (JBC (2010) 285, 38811-38817, Eur Biophys J(2012) 41:959-968

Cultured cells of the blood-brain barrier from apolipoprotein B-100 transgenic mice: effects of oxidized low-density lipoprotein treatment

BACKGROUND: The apolipoprotein B-100 (ApoB-100) transgenic mouse line is a model of human atherosclerosis. Latest findings suggest the importance of ApoB-100 in the development of neurodegenerative diseases and microvascular/perivascular localization of ApoB-100 protein was demonstrated in the cerebral cortex of ApoB-100 transgenic mice. The aim of the study was to characterize cultured brain endothelial cells, pericytes and glial cells from wild-type and ApoB-100 transgenic mice and to study the effect of oxidized low-density lipoprotein (oxLDL) on these cells. METHODS: Morphology of cells isolated from brains of wild type and ApoB-100 transgenic mice was characterized by immunohistochemistry and the intensity of immunolabeling was quantified by image analysis. Toxicity of oxLDL treatment was monitored by real-time impedance measurement and lactate dehydrogenase release. Reactive oxygen species and nitric oxide production, barrier permeability in triple co-culture blood–brain barrier model and membrane fluidity were also determined after low-density lipoprotein (LDL) or oxLDL treatment. RESULTS: The presence of ApoB-100 was confirmed in brain endothelial cells, while no morphological change was observed between wild type and transgenic cells. Oxidized but not native LDL exerted dose-dependent toxicity in all three cell types, induced barrier dysfunction and increased reactive oxygen species (ROS) production in both genotypes. A partial protection from oxLDL toxicity was seen in brain endothelial and glial cells from ApoB-100 transgenic mice. Increased membrane rigidity was measured in brain endothelial cells from ApoB-100 transgenic mice and in LDL or oxLDL treated wild type cells. CONCLUSION: The morphological and functional properties of cultured brain endothelial cells, pericytes and glial cells from ApoB-100 transgenic mice were characterized and compared to wild type cells for the first time. The membrane fluidity changes in ApoB-100 transgenic cells related to brain microvasculature indicate alterations in lipid composition which may be linked to the partial protection against oxLDL toxicity. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12987-015-0013-y) contains supplementary material, which is available to authorized users

Sucrose esters increase drug penetration, but do not inhibit P-glycoprotein in Caco-2 intestinal epithelial cells

Sucrose fatty acid esters are increasingly used as excipients in pharmaceutical products, but few data are available on their toxicity profile, mode of action, and efficacy on intestinal epithelial models. Three water-soluble sucrose esters, palmitate (P-1695), myristate (M-1695), laurate (D-1216), and two reference absorption enhancers, Tween 80 and Cremophor RH40, were tested on Caco-2 cells. Caco-2 monolayers formed a good barrier as reflected by high transepithelial resistance and positive immunostaining for junctional proteins claudin-1, ZO-1, and -catenin. Sucrose esters in nontoxic concentrations significantly reduced resistance and impedance, and increased permeability for atenolol, fluorescein, vinblastine, and rhodamine 123 in Caco-2 monolayers. No visible opening of the tight junctions was induced by sucrose esters assessed by immunohistochemistry and electron microscopy, but some alterations were seen in the structure of filamentous actin microfilaments. Sucrose esters fluidized the plasma membrane and enhanced the accumulation of efflux transporter ligands rhodamine 123 and calcein AM in epithelial cells, but did not inhibit the P-glycoprotein (P- gp)-mediated calcein AM accumulation in MES-SA/Dx5 cell line. These data indicate that in addition to their dissolution-increasing properties sucrose esters can enhance drug permeability through both the transcellular and paracellular routes without inhibiting P-gp

Membrane-Lipid Therapy in Operation: The HSP Co-Inducer BGP-15 Activates Stress Signal Transduction Pathways by Remodeling Plasma Membrane Rafts

Aging and pathophysiological conditions are linked to membrane changes which modulate membrane-controlled molecular switches, causing dysregulated heat shock protein (HSP) expression. HSP co-inducer hydroxylamines such as BGP-15 provide advanced therapeutic candidates for many diseases since they preferentially affect stressed cells and are unlikely have major side effects. In the present study in vitro molecular dynamic simulation, experiments with lipid monolayers and in vivo ultrasensitive fluorescence microscopy showed that BGP-15 alters the organization of cholesterol-rich membrane domains. Imaging of nanoscopic long-lived platforms using the raft marker glycosylphosphatidylinositol-anchored monomeric green fluorescent protein diffusing in the live Chinese hamster ovary (CHO) cell plasma membrane demonstrated that BGP-15 prevents the transient structural disintegration of rafts induced by fever-type heat stress. Moreover, BGP-15 was able to remodel cholesterol-enriched lipid platforms reminiscent of those observed earlier following non-lethal heat priming or membrane stress, and were shown to be obligate for the generation and transmission of stress signals. BGP-15 activation of HSP expression in B16-F10 mouse melanoma cells involves the Rac1 signaling cascade in accordance with the previous observation that cholesterol affects the targeting of Rac1 to membranes. Finally, in a human embryonic kidney cell line we demonstrate that BGP-15 is able to inhibit the rapid heat shock factor 1 (HSF1) acetylation monitored during the early phase of heat stress, thereby promoting a prolonged duration of HSF1 binding to heat shock elements. Taken together, our results indicate that BGP-15 has the potential to become a new class of pharmaceuticals for use in ‘membrane-lipid therapy’ to combat many various protein-misfolding diseases associated with aging

Abstracts from the 20th International Symposium on Signal Transduction at the Blood-Brain Barriers

https://deepblue.lib.umich.edu/bitstream/2027.42/138963/1/12987_2017_Article_71.pd

Poly-(amino acid) polyelectrolyte films: Structure and interactions with proteins and lipids

Summary Following layer-by-layer build-up of poly-(L-glutamic acid)/poly-(L-lysine) (PGA/PLL) multilayers, we could demonstrate the gradual formation of a strong b secondary structure in the polyelectrolyte films. When the chemically very similar poly-(Laspartic acid) (PAA) was used as polyanion, the secondary structure of the polypeptide film was markedly different, it contained sizable amount of a-helix, and random structure as well. PGA and PAA could substitute each other in the polyelectrolyte films, but the structural consequences of the substitutions were not symmetrical: While PAA could be incorporated without large effects, the incorporation of PGA into an existing PAA/PLL film caused major structural rearrangement. The different effects can be related to the longer side chain in the glutamic acid, which results loosened polyelectrolyte film structure. We could create a lipid bilayer on the surface of PGA/PLL polyelectrolyte films. In addition, these lipid bilayers could be 10 covered with another polyelectrolyte layers, thus the lipid double layer was embedded into the polyelectrolyte architecture. This system may provide a tool to incorporate lipid-soluble, hydrophobic compounds into the highly charged polyelectrolyte films, which would be important for practical applications. Moreover, such a lipid double layer may be considered as a new model membrane system, where protein-lipid and protein-membrane interactions can be studied. The underlying polypeptide polyelectrolytes provide a large-scale protein-like surface, thus they can mimic the cytoskeleton, the protein network, which stabilizes the cell membrane. Finally, we made a first attempt to check, whether such artificial lipid bilayers can affect the activity of bio-functionalized polypeptide surfaces. PGA/PLL films, functionalized by a covalently bound melanocyte-stimulating hormone (a-MSH) were covered with lipid double layers. The melanoma cells coming in contact with these surfaces produced somewhat more melanin when the polyelectrolyte architecture contained the lipid double layer as well. These preliminary experiments show, that upon optimization of the lipid composition, and the polyelectrolyte architecture for each intended application, such systems might have practical use in solving biomedical problem

Influence of the Ball Milling Process and Air Sintering Conditions on the Synthesis of La0.7Sr0.3MnO3 Ceramics

Conventional solid-state synthesis was used to produce mixed valence manganite La0.7Ca0.3MnO3 (LCMO) from the mechanochemically activated mixture of the corresponding metal oxides. Prepared samples were characterized by XRD and SEM measurements. The results showed that it is possible to produce single phase LCMO perovskite after at least 2h of ball milling of the reaction mixture, followed by 1400 degrees C sintering of the dry-pressed sample pellets. The prolonged milling time as well as the higher sintering temperature leads to further stabilization of crystal structure

Involvement of small heat shock proteins, trehalose, and lipids in the thermal stress management in Schizosaccharomyces pombe

Changes in the levels of three structurally and functionally different important thermoprotectant molecules, namely small heat shock proteins (sHsps), trehalose, and lipids, have been investigated upon heat shock in Schizosaccharomyces pombe. Both alpha-crystallin-type sHsps (Hsp15.8 and Hsp16) were induced after prolonged high-temperature treatment but with different kinetic profiles. The shsp null mutants display a weak, but significant, heat sensitivity indicating their importance in the thermal stress management. The heat induction of sHsps is different in wild type and in highly heat-sensitive trehalose-deficient (tps1Delta) cells; however, trehalose level did not show significant alteration in shsp mutants. The altered timing of trehalose accumulation and induction of sHsps suggest that the disaccharide might provide protection at the early stage of the heat stress while elevated amount of sHsps are required at the later phase. The cellular lipid compositions of two different temperature-adapted wild-type S. pombe cells are also altered according to the rule of homeoviscous adaptation, indicating their crucial role in adapting to the environmental temperature changes. Both Hsp15.8 and Hsp16 are able to bind to different lipids isolated from S. pombe, whose interaction might provide a powerful protection against heat-induced damages of the membranes. Our data suggest that all the three investigated thermoprotectant macromolecules play a pivotal role during the thermal stress management in the fission yeast

Niosomes decorated with dual ligands targeting brain endothelial transporters increase cargo penetration across the blood-brain barrier

Nanoparticles targeting transporters of the blood-brain barrier (BBB) are promising candidates to increase the brain penetration of biopharmacons. Solute carriers (SLC) are expressed at high levels in brain endothelial cells and show a specific pattern at the BBB. The aim of our study was to test glutathione and ligands of SLC transporters as single or dual BBB targeting molecules for nanovesicles. High mRNA expression levels for hexose and neutral amino acid transporting SLCs were found in isolated rat brain microvessels and our rat primary cell based co-culture BBB model. Niosomes were derivatized with glutathione and SLC ligands glucopyranose and alanine. Serum albumin complexed with Evans blue (67kDa), which has a very low BBB penetration, was selected as a cargo. The presence of targeting ligands on niosomes, especially dual labeling, increased the uptake of the cargo molecule in cultured brain endothelial cells. This cellular uptake was temperature dependent and could be decreased with a metabolic inhibitor and endocytosis blockers filipin and cytochalasin D. Making the negative surface charge of brain endothelial cells more positive with a cationic lipid or digesting the glycocalyx with neuraminidase elevated the uptake of the cargo after treatment with targeted nanocarriers. Treatment with niosomes increased plasma membrane fluidity, suggesting the fusion of nanovesicles with endothelial cell membranes. Targeting ligands elevated the permeability of the cargo across the BBB in the culture model and in mice, and dual-ligand decoration of niosomes was more effective than single ligand labeling. Our data indicate that dual labeling with ligands of multiple SLC transporters can potentially be exploited for BBB targeting of nanoparticles

Novel Engineered Ion Channel Provides Controllable Ion Permeability for Polyelectrolyte Microcapsules Coated with a Lipid Membrane

The development of nanostructured microcapsules based on a biomimetic lipid bilayer membrane (BLM) coating of poly(sodium styrenesulfonate) (PSS)/poly(allylamine hydrochloride) (PAH) polyelectrolyte hollow microcapsules Is reported. A novel engineered ion channel, gramicidin (bis-gA), incorporated Into the lipid membrane coating provides a functional capability to control transport across the microcapsule wall. The microcapsules provide transport and permeation for drug-analog neutral species, as well as positively and negatively charged ionic species. This controlled transport can be tuned for selective release biomimetically by controlling the gating of incorporated bis-gA ion channels. This system provides a platform for the creation of "smart" biomimetic delivery vessels for the effective and selective therapeutic delivery and targeting of drugs