PURPOSE: Dysfunction of the retinal pigment epithelium (RPE) causes numerous forms of retinal degeneration. RPE replacement is a modern option to save vision. We aimed to test the results of transplanting cultured RPEs on biocompatible membranes. METHODS: We cultivated porcine primary RPE cells isolated from cadaver eyes from the slaughterhouse on two types of membranes: commercial polyester scaffolds Transwell (Corning Inc., Kenneburg, ME, USA) with 0.4 μm pore size and prepared Poly (L-lactide-co-DL-lactide) (PDLLA) nanofibrous membranes with an average pore size of 0.4 μm. RESULTS: Five types of assays were used for the analysis: immunocytochemistry (ICC), phagocytosis assay, Western blotting, real-time qPCR (RT-qPCR) and electron microscopy. RT-qPCR demonstrated that RPEs cultured on nanofibrous membranes have higher expressions of BEST1 (bestrophin 1), RLBP1 (retinaldehyde-binding protein 1), RPE65 (retinal pigment epithelium-specific 65 kDa protein), PAX6 (transcription factor PAX6), SOX9 (transcription factor SOX9), DCT (dopachrome tautomerase) and MITF (microphthalmia-associated transcription factor). ICC of the RPEs cultured on nanofibrous membranes showed more intensive staining of markers such as BEST1, MCT1 (monocarboxylate transporter 1), Na+ /K+ ATPase, RPE65 and acetylated tubulin in comparison with commercial ones. Additionally, the absence of α-SMA proved the stability of the RPE polarization state and the absence of epithelial-to-mesenchymal transition. RPE possessed high phagocytic activity. Electron microscopy of both membranes confirmed a confluent layer of RPE cells and their genuine morphological structure, which was comparable to native RPEs. CONCLUSIONS: Retinal pigment epitheliums cultured on polylactide nanofibrous membranes improved the final quality of the cell product by having better maturation and long-term survival of the RPE monolayer compared to those cultured on commercial polyester scaffolds. PDLLA-cultured RPEs are a plausible source for the replacement of non-functioning RPEs during cell therapy.

• MeSH
• Bestrophins metabolism   MeSH
• Retinal Degeneration * metabolism   MeSH
• Cells, Cultured MeSH
• Nanofibers * chemistry   MeSH
• Polyesters metabolism   MeSH
• Swine MeSH
• Retinal Pigment Epithelium metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

PURPOSE: The tear film lipid layer (TFLL) covers the tear film, stabilizing it and providing a protective barrier against the environment. The TFLL is divided into polar and non-polar sublayers, but the interplay between lipid classes in these sublayers and the structure-function relationship of the TFLL remains poorly characterized. This study aims to provide insight into TFLL function by elucidating the interactions between polar and non-polar TFLL lipids at the molecular level. METHODS: Mixed films of polar O-acyl-ω-hydroxy fatty acids (OAHFA) or phospholipids and non-polar cholesteryl esters (CE) were used as a model of the TFLL. The organization of the films was studied by using a combination of Brewster angle and fluorescence microscopy in a Langmuir trough system. In addition, the evaporation resistance of the lipid films was evaluated. RESULTS: Phospholipids and OAHFAs induced the formation of a stable multilamellar CE film. The formation of this film was driven by the interdigitation of acyl chains between the monolayer of polar lipids and the CE multilayer lamellae. Surprisingly, the multilayer structure was destabilized by both low and high concentrations of polar lipids. In addition, the CE multilayer was no more effective in resisting the evaporation of water than a polar lipid monolayer. CONCLUSIONS: Formation of multilamellar films by major tear film lipids suggest that the TFLL may have a similar structure. Moreover, in contrast to the current understanding, polar TFLL lipids may not mainly act by stabilizing the non-polar TFLL sublayer, but through a direct evaporation resistant effect.

• MeSH
• Cholesterol Esters MeSH
• Lipids MeSH
• Fatty Acids MeSH
• Tears * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.

• MeSH
• Actins drug effects   metabolism   MeSH
• Cell Adhesion MeSH
• Antigens, CD drug effects   metabolism   MeSH
• Endothelium, Vascular MeSH
• Human Umbilical Vein Endothelial Cells MeSH
• Endothelial Cells drug effects   metabolism   physiology   MeSH
• Neovascularization, Physiologic drug effects   physiology   MeSH
• Cadherins drug effects   metabolism   MeSH
• Actin-Related Protein 2-3 Complex metabolism   MeSH
• Myosin Light Chains metabolism   MeSH
• Humans MeSH
• Intercellular Junctions drug effects   metabolism   MeSH
• Microtubules drug effects   metabolism   MeSH
• Models, Cardiovascular MeSH
• Cell Movement drug effects   physiology   MeSH
• Cell Polarity drug effects   physiology   MeSH
• Actin-Related Protein 2 metabolism   MeSH
• Actin-Related Protein 3 metabolism   MeSH
• Wiskott-Aldrich Syndrome Protein metabolism   MeSH
• Pseudopodia drug effects   metabolism   physiology   MeSH
• rac GTP-Binding Proteins metabolism   MeSH
• Vascular Endothelial Growth Factor Receptor-2 metabolism   MeSH
• Vascular Remodeling MeSH
• Wiskott-Aldrich Syndrome Protein Family metabolism   MeSH
• Signal Transduction MeSH
• Cardiac Myosins metabolism   MeSH
• Vascular Endothelial Growth Factor A metabolism   pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Mitochondria are crucial compartments of eukaryotic cells because they function as the cellular power plant and play a central role in the early stages of programmed cell death (apoptosis). To avoid undesired cell death, this apoptotic pathway is tightly regulated by members of the Bcl-2 protein family, which interact on the external surface of the mitochondria, i.e., the mitochondrial outer membrane (MOM), and modulate its permeability to apoptotic factors, controlling their release into the cytosol. A growing body of evidence suggests that the MOM lipids play active roles in this permeabilization process. In particular, oxidized phospholipids (OxPls) formed under intracellular stress seem to directly induce apoptotic activity at the MOM. Here we show that the process of MOM pore formation is sensitive to the type of OxPls species that are generated. We created MOM-mimicking liposome systems, which resemble the cellular situation before apoptosis and upon triggering of oxidative stress conditions. These vesicles were studied using (31)P solid-state magic-angle-spinning nuclear magnetic resonance spectroscopy and differential scanning calorimetry, together with dye leakage assays. Direct polarization and cross-polarization nuclear magnetic resonance experiments enabled us to probe the heterogeneity of these membranes and their associated molecular dynamics. The addition of apoptotic Bax protein to OxPls-containing vesicles drastically changed the membranes' dynamic behavior, almost completely negating the previously observed effect of temperature on the lipids' molecular dynamics and inducing an ordering effect that led to more cooperative membrane melting. Our results support the hypothesis that the mitochondrion-specific lipid cardiolipin functions as a first contact site for Bax during its translocation to the MOM in the onset of apoptosis. In addition, dye leakage assays revealed that different OxPls species in the MOM-mimicking vesicles can have opposing effects on Bax pore formation.

• MeSH
• Apoptosis physiology   MeSH
• Calorimetry, Differential Scanning MeSH
• Escherichia coli MeSH
• Fluorescent Dyes MeSH
• Phospholipids metabolism   MeSH
• Cardiolipins metabolism   MeSH
• Humans MeSH
• Lipid Bilayers chemistry   MeSH
• Mitochondrial Membranes metabolism   MeSH
• Mitochondria metabolism   MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress physiology   MeSH
• Cell Membrane Permeability MeSH
• bcl-2-Associated X Protein metabolism   MeSH
• Temperature MeSH
• Unilamellar Liposomes chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Steroids constitute one of the most important groups of compounds of regulatory properties both in the animal and plant kingdom. In plants, steroids such as brassinosteroids or progesterone, by binding to protein receptors in cell membranes, regulate growth and initiate processes leading to increased tolerance to stress conditions. Due to their structural similarities to sterols, these steroids may also directly interact with cellular membranes. Our aim was to determine the changes of the structural parameters of lipid membranes under the influence of hydrophobic steroid compounds, i.e., 24-epibrassinolide (EBR) and its precursor-24-epicastasterone (ECS) and progesterone (PRO). Lipids were isolated from wheat seedlings with different tolerances to frost, grown at low temperatures (5 °C) for 1.5 and 3 weeks (acclimation process). Control plants were cultured continuously at 20 °C. From galactolipids and phospholipids, the main polar lipid fractions, the monolayers were formed, using a technique of Langmuir trough. EBR and ECS were introduced into monolayers, together with lipids, whereas the PRO was dissolved in the aqueous sub-phase upon which the monolayers were spread. Measurements performed at 25 °C and 10 °C showed a significant action of the tested compounds on the physicochemical properties of the monolayers. EBR and PRO increased the area per lipid molecule in monolayers, resulting in formation of more flexible surface structures while the presence of the ECS induced the opposite effect. The influence of the polarity of lipids and steroids on the interactions in the monolayer was discussed. Lipids extracted from the membranes of wheat with the most tolerance to frost were characterized by the highest fatty acid unsaturation and steroids had a relatively weak effect on the parameters of the structure of their monolayers.

• MeSH
• Brassinosteroids chemistry   metabolism   pharmacology   MeSH
• Cell Membrane chemistry   drug effects   MeSH
• Membrane Lipids chemistry   metabolism   MeSH
• Lipid Metabolism drug effects   MeSH
• Cold Temperature MeSH
• Progesterone chemistry   metabolism   pharmacology   MeSH
• Triticum drug effects   growth & development   MeSH
• Seedlings drug effects   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

1. Emtricitabine is a nucleoside reverse transcriptase inhibitor used in combination antiretroviral therapy of HIV (cART). Although active transport mechanisms are believed to mediate tubular secretion of the drug into urine, the responsible transporter and its potential to cause pharmacokinetic drug--drug interactions (DDI) has not been identified so far. The aim of this study was to investigate whether drug transporters P-gp (ABCB1), BCRP (ABCG2), MRP2 (ABCC2), OCT1 (SLC22A1), OCT2 (SLC22A2) or MATE1 (SLC47A1) can mediate active transcellular transfer of emtricitabine. 2. We employed transport assays in polarized monolayers of MDCK cells stably expressing P-gp, BCRP, MRP2, OCT1, OCT2 and/or MATE1. Among the transporters studied only MATE1 accelerated basal-to-apical transport of emtricitabine over a wide range of concentrations (6 nM to 1 mM). The transport was enhanced by an oppositely directed pH gradient and significantly reduced (p < 0.001) at low temperature in MDCK-MATE1, MDCK-OCT1/MATE1 and MDCK-OCT2/MATE1 cells. Co-administration of cimetidine or ritonavir decreased MATE1-mediated transport of emtricitabine by up to 42 and 39%, respectively (p < 0.01) and augmented intracellular accumulation of emtricitabine (p < 0.05). 3. We demonstrate emtricitabine as a substrate of MATE1 and suggest that MATE1 might cause DDI between emtricitabine and other co-administrated drugs including antiretrovirals.

• MeSH
• Madin Darby Canine Kidney Cells MeSH
• Emtricitabine metabolism   MeSH
• Reverse Transcriptase Inhibitors metabolism   MeSH
• ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism   MeSH
• Organic Cation Transport Proteins metabolism   MeSH
• Dogs MeSH
• Animals MeSH
• Check Tag
• Dogs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

• MeSH
• Cell Membrane metabolism   MeSH
• Phospholipids chemistry   metabolism   MeSH
• Lipid Bilayers chemistry   metabolism   MeSH
• Liposomes chemistry   metabolism   MeSH
• Models, Molecular MeSH
• Molecular Dynamics Simulation MeSH
• Calcium metabolism   MeSH
• Calcium Signaling MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface.

• MeSH
• Calorimetry, Differential Scanning MeSH
• Phosphorylcholine analogs & derivatives   metabolism   MeSH
• Humans MeSH
• Carbon-13 Magnetic Resonance Spectroscopy MeSH
• Mitochondrial Membranes metabolism   MeSH
• Oxidation-Reduction MeSH
• Permeability MeSH
• bcl-2-Associated X Protein metabolism   MeSH
• Proton Magnetic Resonance Spectroscopy MeSH
• Unilamellar Liposomes MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Cells * MeSH
• Molecular Biology MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• molekulární biologie, molekulární medicína
• NML Publication type
• učebnice vysokých škol

• MeSH
• Chemistry, Physical MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Fyzikální chemie
• NML Fields
• chemie, klinická chemie