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Reconstituted lipid vesicles

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https://www.readbyqxmd.com/read/28912594/quantification-of-aquaporin-z-reconstituted-into-vesicles-for-biomimetic-membrane-fabrication
#1
Hui Xian Gan, Hu Zhou, Qingsong Lin, Yen Wah Tong
Aquaporin incorporated biomimetic membranes are anticipated to offer unprecedented desalination capabilities. However, the lack of accurate methods to quantify the reconstituted aquaporin presents a huge hurdle in investigating aquaporin performance and optimizing membrane fabrication. Herein, we present three quantification methods to determine the Aquaporin-Z reconstituted into E. coli lipid vesicles: 1) nanogold labeling with transmission electron microscopy (TEM) visualization, 2) nickel labeling with inductively coupled plasma-mass spectrometry (ICP-MS) and 3) gel electrophoresis...
September 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28858288/simultaneous-lipid-and-content-mixing-assays-for-in-vitro-reconstitution-studies-of-synaptic-vesicle-fusion
#2
Xiaoxia Liu, Alpay Burak Seven, Junjie Xu, Victoria Esser, Lijing Su, Cong Ma, Josep Rizo
This protocol describes reconstitution assays to study how the neurotransmitter release machinery triggers Ca(2+)-dependent synaptic vesicle fusion. The assays monitor fusion between proteoliposomes containing the synaptic vesicle SNARE synaptobrevin (with or without the Ca(2+) sensor synaptotagmin-1) and proteoliposomes initially containing the plasma membrane SNAREs syntaxin-1 and soluble NSF attachment protein (SNAP)-25. Lipid mixing (from fluorescence de-quenching of Marina-Blue-labeled lipids) and content mixing (from development of fluorescence resonance energy transfer (FRET) between phycoerythrin-biotin (PhycoE-Biotin) and Cy5-streptavidin trapped in the two proteoliposome populations) are measured simultaneously to ensure that true, nonleaky membrane fusion is monitored...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28854360/spatiotemporal-control-of-lipid-conversion-actin-based-mechanical-forces-and-curvature-sensors-during-clathrin-ap-1-coated-vesicle-biogenesis
#3
Mihaela Anitei, Christoph Stange, Cornelia Czupalla, Christian Niehage, Kai Schuhmann, Pia Sala, Aleksander Czogalla, Theresia Pursche, Ünal Coskun, Andrej Shevchenko, Bernard Hoflack
Clathrin/adaptor protein-1-coated carriers connect the secretory and the endocytic pathways. Carrier biogenesis relies on distinct protein networks changing membrane shape at the trans-Golgi network, each regulating coat assembly, F-actin-based mechanical forces, or the biophysical properties of lipid bilayers. How these different hubs are spatiotemporally coordinated remains largely unknown. Using in vitro reconstitution systems, quantitative proteomics, and lipidomics, as well as in vivo cell-based assays, we characterize the protein networks controlling membrane lipid composition, membrane shape, and carrier scission...
August 29, 2017: Cell Reports
https://www.readbyqxmd.com/read/28842688/light-independent-phospholipid-scramblase-activity-of-bacteriorhodopsin-from-halobacterium-salinarum
#4
Alice Verchère, Wei-Lin Ou, Birgit Ploier, Takefumi Morizumi, Michael A Goren, Peter Bütikofer, Oliver P Ernst, George Khelashvili, Anant K Menon
The retinylidene protein bacteriorhodopsin (BR) is a heptahelical light-dependent proton pump found in the purple membrane of the archaeon Halobacterium salinarum. We now show that when reconstituted into large unilamellar vesicles, purified BR trimers exhibit light-independent lipid scramblase activity, thereby facilitating transbilayer exchange of phospholipids between the leaflets of the vesicle membrane at a rate >10,000 per trimer per second. This activity is comparable to that of recently described scramblases including bovine rhodopsin and fungal TMEM16 proteins...
August 25, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28827281/munc18a-clusters-snare-bearing-liposomes-prior-to-trans-snare-zippering
#5
Matthew Grant Arnold, Pratikshya Adhikari, Baobin Kang, Hao Xu
Sec1-Munc18 (SM) proteins cooperate with SNAREs {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein] receptors} to mediate membrane fusion in eukaryotic cells. Studies of Munc18a/Munc18-1/Stxbp1 in neurotransmission suggest that SM proteins accelerate fusion kinetics primarily by activating the partially zippered trans-SNARE complex. However, accumulating evidence has argued for additional roles for SM proteins in earlier steps in the fusion cascade. Here we investigate the function of Munc18a in reconstituted exocytic reactions mediated by neuronal and non-neuronal SNAREs...
August 21, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/28822127/in-vitro-analysis-of-metabolite-transport-proteins
#6
Marc-Sven Roell, Franziska Kuhnert, Shirin Zamani-Nour, Andreas P M Weber
The photorespiratory cycle is distributed over four cellular compartments, the chloroplast, peroxisomes, cytoplasm, and mitochondria. Shuttling of photorespiratory intermediates between these compartments is essential to maintain the function of photorespiration. Specific transport proteins mediate the transport across biological membranes and represent important components of the cellular metabolism. Although significant progress was made in the last years on identifying and characterizing new transport proteins, the overall picture of intracellular metabolite transporters is still rather incomplete...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28768431/adefovir-dipivoxil-loaded-proliposomal-powders-with-improved-hepatoprotective-activity-formulation-optimization-pharmacokinetic-and-biodistribution-studies
#7
Ghada A Abdelbary, Maha M Amin, Mohamed Y Zakaria, Sally A El Awdan
The present study aimed to prepare proliposomal formulae for improving the oral bioavailability of Adefovir Dipivoxil (AD), a nucleoside reverse transcriptase inhibitor effective against Hepatitis B virus (HBV). The prepared proliposomal formulae were characterized for entrapment efficiency (E.E.%), vesicle size and in-vitro drug release after reconstitution to conventional liposomes. The optimized formula (F9) with a maximum desirability value of 0.858 was selected having E.E.% of 71±3.3% with an average vesicle size of 164...
August 2, 2017: Journal of Liposome Research
https://www.readbyqxmd.com/read/28703789/capturing-suboptical-dynamic-structures-in-lipid-bilayer-patches-formed-from-free-standing-giant-unilamellar-vesicles
#8
Tripta Bhatia, Flemming Cornelius, John H Ipsen
There is accumulating evidence that the small-scale lateral organization of biological membranes has a crucial role in signaling and trafficking in cells. However, it has been difficult to characterize these features with existing methods for preparing and analyzing freestanding membranes, because the dynamics occurs below the optical resolution possible with these protocols. We have developed a protocol that permits the imaging of lipid nanodomains and lateral protein organization in membranes of giant unilamellar vesicles (GUVs)...
August 2017: Nature Protocols
https://www.readbyqxmd.com/read/28697898/fusion-domains-guide-the-oriented-insertion-of-light-driven-proton-pumps-into-liposomes
#9
Noah Ritzmann, Johannes Thoma, Stephan Hirschi, David Kalbermatter, Dimitrios Fotiadis, Daniel J Müller
One major objective of synthetic biology is the bottom-up assembly of minimalistic nanocells consisting of lipid or polymer vesicles as architectural scaffolds and of membrane and soluble proteins as functional elements. However, there is no reliable method to orient membrane proteins reconstituted into vesicles. Here, we introduce a simple approach to orient the insertion of the light-driven proton pump proteorhodopsin (PR) into liposomes. To this end, we engineered red or green fluorescent proteins to the N- or C-terminus of PR, respectively...
July 8, 2017: Biophysical Journal
https://www.readbyqxmd.com/read/28695207/peripheral-myelin-protein-22-alters-membrane-architecture
#10
Kathleen F Mittendorf, Justin T Marinko, Cheri M Hampton, Zunlong Ke, Arina Hadziselimovic, Jonathan P Schlebach, Cheryl L Law, Jun Li, Elizabeth R Wright, Charles R Sanders, Melanie D Ohi
Peripheral myelin protein 22 (PMP22) is highly expressed in myelinating Schwann cells of the peripheral nervous system. PMP22 genetic alterations cause the most common forms of Charcot-Marie-Tooth disease (CMTD), which is characterized by severe dysmyelination in the peripheral nerves. However, the functions of PMP22 in Schwann cell membranes remain unclear. We demonstrate that reconstitution of purified PMP22 into lipid vesicles results in the formation of compressed and cylindrically wrapped protein-lipid vesicles that share common organizational traits with compact myelin of peripheral nerves in vivo...
July 2017: Science Advances
https://www.readbyqxmd.com/read/28688734/first-moves-towards-photoautotrophic-synthetic-cells-in-vitro-study-of-photosynthetic-reaction-centre-and-cytochrome-bc1-complex-interactions
#11
Emiliano Altamura, Rosa Fiorentino, Francesco Milano, Massimo Trotta, Gerardo Palazzo, Pasquale Stano, Fabio Mavelli
Following a bottom-up synthetic biology approach it is shown that vesicle-based cell-like systems (shortly "synthetic cells") can be designed and assembled to perform specific function (for biotechnological applications) and for studies in the origin-of-life field. We recently focused on the construction of synthetic cells capable to converting light into chemical energy. Here we first present our approach, which has been realized so far by the reconstitution of photosynthetic reaction centre in the membrane of giant lipid vesicles...
June 26, 2017: Biophysical Chemistry
https://www.readbyqxmd.com/read/28648851/the-influence-of-mild-acidity-on-lysyl-phosphatidylglycerol-biosynthesis-and-lipid-membrane-physico-chemical-properties-in-methicillin-resistant-staphylococcus-aureus
#12
Reg P Rehal, Helene Marbach, Alasdair T M Hubbard, Anam A Sacranie, Federica Sebastiani, Giovanna Fragneto, Richard D Harvey
The increased biosynthesis of lysyl-phosphatidylglycerol in Staphylococcus aureus when cultured under conditions of mild acidity and the resultant increased proportion of this lipid in the plasma membrane of the bacterium, alters the physico-chemical properties of lipid bilayers in a manner which is itself dependent upon environmental pH. Clinically relevant strains of S. aureus, both methicillin susceptible and resistant, all exhibited increased lysyl-phosphatidylglycerol biosynthesis in response to mild environmental acidity, albeit to differing degrees, from ∼30% to ∼55% total phospholipid...
August 2017: Chemistry and Physics of Lipids
https://www.readbyqxmd.com/read/28616949/monitoring-atpase-induced-ph-changes-in-single-proteoliposomes-with-the-lipid-coupled-fluorophore-oregon-green-488
#13
Miriam Schwamborn, Johannes Schumacher, Jeremias Sibold, Nikolas K Teiwes, Claudia Steinem
Monitoring the proton pumping activity of proteins such as ATPases in reconstituted single proteoliposomes is key to quantify the function of proteins as well as potential proton pump inhibitors. However, most pH-detecting assays available are either not quantitative, require well-adapted reconstitution protocols or are not appropriate for single vesicle studies. Here, we describe the quantitative and time-resolved detection of F-type ATPase-induced pH changes across vesicular membranes doped with the commercially available pH sensitive fluorophore Oregon Green 488 DHPE...
July 10, 2017: Analyst
https://www.readbyqxmd.com/read/28592883/the-lateral-distance-between-a-proton-pump-and-atp-synthase-determines-the-atp-synthesis-rate
#14
Johannes Sjöholm, Jan Bergstrand, Tobias Nilsson, Radek Šachl, Christoph von Ballmoos, Jerker Widengren, Peter Brzezinski
We have investigated the effect of lipid composition on interactions between cytochrome bo 3 and ATP-synthase, and the ATP-synthesis activity driven by proton pumping. The two proteins were labeled by fluorescent probes and co-reconstituted in large (d ≅ 100 nm) or giant (d ≅ 10 µm) unilamellar lipid vesicles. Interactions were investigated using fluorescence correlation/cross-correlation spectroscopy and the activity was determined by measuring ATP production, driven by electron-proton transfer, as a function of time...
June 7, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28536532/phase-partitioning-of-gm1-and-its-bodipy-labeled-analog-determine-their-different-binding-to-cholera-toxin
#15
Sami Rissanen, Michal Grzybek, Adam Orłowski, Tomasz Róg, Oana Cramariuc, Ilya Levental, Christian Eggeling, Erdinc Sezgin, Ilpo Vattulainen
Driven by interactions between lipids and proteins, biological membranes display lateral heterogeneity that manifests itself in a mosaic of liquid-ordered (Lo) or raft, and liquid-disordered (Ld) or non-raft domains with a wide range of different properties and compositions. In giant plasma membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB) to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1), whose headgroup accessibility and membrane order or phase partitioning may differ from those of GM1, rendering the interpretation of CTxB binding data quite problematic...
2017: Frontiers in Physiology
https://www.readbyqxmd.com/read/28476622/green-fluorescence-protein-based-content-mixing-assay-of-snare-driven-membrane-fusion
#16
Paul Heo, Byoungjae Kong, Young-Hun Jung, Joon-Bum Park, Jonghyeok Shin, Myungseo Park, Dae-Hyuk Kweon
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion by forming a ternary SNARE complex. A minimalist approach utilizing proteoliposomes with reconstituted SNARE proteins yielded a wealth of information pinpointing the molecular mechanism of SNARE-mediated fusion and its regulation by accessory proteins. Two important attributes of a membrane fusion are lipid-mixing and the formation of an aqueous passage between apposing membranes. These two attributes are typically observed by using various fluorescent dyes...
June 17, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28472750/effect-of-the-presence-of-cholesterol-in-the-interfacial-microenvironment-on-the-modulation-of-the-alkaline-phosphatase-activity-during-in-vitro-mineralization
#17
B F Favarin, M A R Andrade, M Bolean, A M S Simão, A P Ramos, M F Hoylaerts, J L Millán, P Ciancaglini
Mineralization of the skeleton starts within cell-derived matrix vesicles (MVs); then, minerals propagate to the extracellular collagenous matrix. Tissue-nonspecific alkaline phosphatase (TNAP) degrades inorganic pyrophosphate (PPi), a potent inhibitor of mineralization, and contributes Pi (Phosphate) from ATP to initiate mineralization. Compared to the plasma membrane, MVs are rich in Cholesterol (Chol) (∼32%) and TNAP, but how Chol influences TNAP activity remains unclear. We have reconstituted TNAP in liposomes of dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) combined with Chol or its derivatives Cholestenone (Achol) and Ergosterol (Ergo)...
April 26, 2017: Colloids and Surfaces. B, Biointerfaces
https://www.readbyqxmd.com/read/28415454/development-of-a-dna-liposome-complex-for-gene-delivery-applications
#18
M Rasoulianboroujeni, G Kupgan, F Moghadam, M Tahriri, A Boughdachi, P Khoshkenar, J J Ambrose, N Kiaie, D Vashaee, J D Ramsey, L Tayebi
The association structures formed by cationic liposomes and DNA (Deoxyribonucleic acid)-liposome have been effectively utilized as gene carriers in transfection assays. In this research study, cationic liposomes were prepared using a modified lipid film hydration method consisting of a lyophilization step for gene delivery applications. The obtained results demonstrated that the mean particle size had no significant change while the polydispersity (PDI) increased after lyophilization. The mean particle size slightly reduced after lyophilization (520±12nm to 464±25nm) while the PDI increased after lyophilization (0...
June 1, 2017: Materials Science & Engineering. C, Materials for Biological Applications
https://www.readbyqxmd.com/read/28408867/visualization-of-snare-mediated-hemifusion-between-giant-unilamellar-vesicles-arrested-by-myricetin
#19
Paul Heo, Joon-Bum Park, Yeon-Kyun Shin, Dae-Hyuk Kweon
Neurotransmitters are released within a millisecond after Ca(2+) arrives at an active zone. However, the vesicle fusion pathway underlying this synchronous release is yet to be understood. At the center of controversy is whether hemifusion, in which outer leaflets are merged while inner leaflets are still separated, is an on-pathway or off-pathway product of Ca(2+)-triggered exocytosis. Using the single vesicle fusion assay, we recently demonstrated that hemifusion is an on-pathway intermediate that immediately proceeds to full fusion upon Ca(2+) triggering...
2017: Frontiers in Molecular Neuroscience
https://www.readbyqxmd.com/read/28377464/stard3-mediates-endoplasmic-reticulum-to-endosome-cholesterol-transport-at-membrane-contact-sites
#20
Léa P Wilhelm, Corinne Wendling, Benoît Védie, Toshihide Kobayashi, Marie-Pierre Chenard, Catherine Tomasetto, Guillaume Drin, Fabien Alpy
StAR-related lipid transfer domain-3 (STARD3) is a sterol-binding protein that creates endoplasmic reticulum (ER)-endosome contact sites. How this protein, at the crossroad between sterol uptake and synthesis pathways, impacts the intracellular distribution of this lipid was ill-defined. Here, by using in situ cholesterol labeling and quantification, we demonstrated that STARD3 induces cholesterol accumulation in endosomes at the expense of the plasma membrane. STARD3-mediated cholesterol routing depends both on its lipid transfer activity and its ability to create ER-endosome contacts...
May 15, 2017: EMBO Journal
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