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Lipid Vesicles (lipid + vesicle)
Selected AbstractsAffinity Purification of Lipid VesiclesBIOTECHNOLOGY PROGRESS, Issue 1 2004Boris Peker We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 × 1013 active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 × 108 M,1. We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format. [source] Protein Incorporation in Giant Lipid Vesicles under Physiological ConditionsCHEMBIOCHEM, Issue 2 2010Paige M. Shaklee Dr. Life's construction zone: Proteins were incorporated into giant vesicles (GVs) under physiological conditions by using electroformation. The figure shows these fluorescently labeled GVs in which proteins are encapsulated. Our method opens doors to investigating the membrane properties of native, intracellular membranes. [source] Building artificial cells and protocell models: Experimental approaches with lipid vesiclesBIOESSAYS, Issue 4 2010Peter Walde Abstract Lipid vesicles are often used as compartment structures for preparing cell-like systems and models of protocells, the hypothetical precursor structures of the first cells at the origin of life. Although the various artificially made vesicle systems are already remarkably complex, they are still very different from and much simpler than any known living cell. Nevertheless, the preparation and study of the structure and the dynamics of functionalized vesicle systems may contribute to a better understanding of biological cells, in particular of the essential features of a living cell that are not found in the non-living form of matter. The study of protocell models may possibly lead to a better understanding of the origin of the first cells. To avoid misunderstanding in this field of research, it would be useful if generally accepted definitions of terms like "artificial cells," "synthetic cells," "minimal cells," "protocells," and "primitive cells" exist. , Editor's suggested further reading in BioEssays Synthetic cells and organelles: compartmentalization strategiesAbstract [source] Comparative folliculogenesis and spermatogenesis of four teleost fish from a Reservoir in south-eastern BrazilACTA ZOOLOGICA, Issue 4 2010Yuri Simões Martins Abstract Martins, Y.S., Moura, D.F., Santos, G.B., Rizzo, E. and Bazzoli, N. 2009. Comparative folliculogenesis and spermatogenesis of four teleost fish from a Reservoir in south-eastern Brazil. ,Acta Zoologica (Stockholm). 91: 466,473. This study provides a comparative analysis of gametogenesis of neotropical teleosts Metynnis maculatus, Megalancistrus parananus, Cichla kelberi and Satanoperca pappaterra, through histological, histochemical and histometric techniques. In the ooplasm of C. kelberi and S. pappaterra conspicuous lipid vesicles were observed, which are characteristic of pelagic eggs produced by marine fishes. Perinucleolar oocytes were identified in the testis of S. pappaterra suggesting that this species is protogynous without functional hermaphroditism, an unusual pattern for neotropical fresh-water fishes. The spermatozoa of the studied species have rounded heads, a characteristic of fish that externally fertilise their eggs. The follicular (granulosa) cells of the vitellogenic oocytes from the studied species were cuboidal or columnar, however, in C. kelberi there were columnar follicular cells at the vegetal pole and cuboidal cells at the animal pole. Variations of the histochemical content were detected in the cortical alveoli and follicular cells of vitellogenic oocytes showing differences in the mechanisms to block polyspermy and egg adhesiveness. Larger oocytes were recorded in species which demonstrated parental care behaviour and smaller oocytes were noted in those species with fractioned spawning. [source] Liposomes for entrapping local anesthetics: A liposome electrokinetic chromatographic studyELECTROPHORESIS, Issue 9 2010Jana Lokajová Abstract Bupivacaine is a lipophilic, long-acting, amide class local anesthetic commonly used in clinical practice to provide local anesthesia during surgical procedures. Several cases of accidental overdose with cardiac arrest and death have been reported since bupivacaine was introduced to human use. Recent case reports have suggested that Intralipid (Fresenius Kabi) is an effective therapy for cardiac toxicity from high systemic concentrations of, e.g. bupivacaine, even though the mechanism behind the interaction is not fully clear yet. Our long-term aim is to develop a sensitive, efficient, and non-harmful lipid-based formulation to specifically trap harmful substances in vivo. In this study, the in vitro interaction of local anesthetics (bupivacaine, prilocaine, and lidocaine) with Intralipid or lipid vesicles containing phosphatidylglycerol, phosphatidylcholine, cardiolipin, cholesterol, and N -palmitoyl- D - erythro -sphingosine (ceramide) was determined by liposome electrokinetic chromatography. The interactions were evaluated by calculating the retention factors and distribution constants. Atomic force microscopy measurements were carried out to confirm that the interaction mechanism was solely due to interactions between the analytes and the moving pseudostationary phase and not by interactions with a stationary lipid phase adsorbed to the fused-silica wall. The heterogeneity of the liposomes was also studied by atomic force microscopy. The liposome electrokinetic chromatography results demonstrate that there is higher interaction between the drugs and negatively charged liposome dispersion than with the commercial Intralipid dispersion. [source] Lipid-induced conformational transition of the amyloid core fragment A,(28,35) and its A30G and A30I mutantsFEBS JOURNAL, Issue 10 2008Sureshbabu Nagarajan The interaction of the ,-amyloid peptide (A,) with neuronal membranes could play a key role in the pathogenesis of Alzheimer's disease. Recent studies have focused on the interactions of A, oligomers to explain the neuronal toxicity accompanying Alzheimer's disease. In our study, we have investigated the role of lipid interactions with soluble A,(28,35) (wild-type) and its mutants A30G and A30I in their aggregation and conformational preferences. CD and Trp fluorescence spectroscopic studies indicated that, immediately on dissolution, these peptides adopted a random coil structure. Upon addition of negatively charged 1,2-dipalmitoyl- syn -glycero-3-phospho- rac -(glycerol) sodium salt (PG) lipid, the wild-type and A30I mutant underwent reorganization into a predominant ,-sheet structure. However, no conformational changes were observed in the A30G mutant on interaction with PG. In contrast, the presence of zwitterionic 1,2-dipalmitoyl- syn -glycero-3-phosphatidylcholine (PC) lipid had no effect on the conformation of these three peptides. These observations were also confirmed with atomic force microscopy and the thioflavin-T assay. In the presence of PG vesicles, both the wild-type and A30I mutant formed fibrillar structures within 2 days of incubation in NaCl/Pi, but not in their absence. Again, no oligomerization was observed with PC vesicles. The Trp studies also revealed that both ends of the three peptides are not buried deep in the vesicle membrane. Furthermore, fluorescence spectroscopy using the environment-sensitive probe 1,6-diphenyl-1,3,5-hexatriene showed an increase in the membrane fluidity upon exposure of the vesicles to the peptides. The latter effect may result from the lipid head group interactions with the peptides. Fluorescence resonance energy transfer experiments revealed that these peptides undergo a random coil-to-sheet conversion in solution on aging and that this process is accelerated by negatively charged lipid vesicles. These results indicate that aggregation depends on hydrophobicity and propensity to form ,-sheets of the amyloid peptide, and thus offer new insights into the mechanism of amyloid neurodegenerative disease. [source] Peptides corresponding to helices 5 and 6 of Bax can independently form large lipid poresFEBS JOURNAL, Issue 5 2006Ana J. García-Sáez Proteins of the B-cell lymphoma protein 2 (Bcl2) family are key regulators of the apoptotic cascade, controlling the release of apoptotic factors from the mitochondrial intermembrane space. A helical hairpin found in the core of water-soluble folds of these proteins has been reported to be the pore-forming domain. Here we show that peptides including any of the two ,-helix fragments of the hairpin of Bcl2 associated protein X (Bax) can independently induce release of large labelled dextrans from synthetic lipid vesicles. The permeability promoted by these peptides is influenced by intrinsic monolayer curvature and accompanied by fast transbilayer redistribution of lipids, supporting a toroidal pore mechanism as in the case of the full-length protein. However, compared with the pores made by complete Bax, the pores made by the Bax peptides are smaller and do not need the concerted action of tBid. These data indicate that the sequences of both fragments of the hairpin contain the principal physicochemical requirements for pore formation, showing a parallel between the permeabilization mechanism of a complex regulated protein system, such as Bax, and the much simpler pore-forming antibiotic peptides. [source] Detergent-resistant membranes are platforms for actinoporin pore-forming activity on intact cellsFEBS JOURNAL, Issue 4 2006Jorge Alegre-Cebollada Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus. We studied its cytolytic activity on COS-7 cells. Fluorescence spectroscopy and flow cytometry revealed that the toxin permeabilizes cells to propidium cations in a dose-dependent and time-dependent manner. This permeabilization is impaired by preincubation of cells with cyclodextrin. Isolation of detergent-resistant cellular membranes showed that sticholysin II colocalizes with caveolin-1 in fractions corresponding to raft-like domains. The interaction of sticholysin II with such domains is only lipid dependent as it also occurs in the absence of any other membrane-associated protein. Toxin binding to raft-like lipid vesicles inhibited cell permeabilization. The results suggest that sticholysin II promotes pore formation in COS-7 cells through interaction with membrane domains which behave like cellular rafts. [source] Interaction of ostreolysin, a cytolytic protein from the edible mushroom Pleurotus ostreatus, with lipid membranes and modulation by lysophospholipidsFEBS JOURNAL, Issue 6 2003Kristina Sep Ostreolysin is a 16-kDa cytolytic protein specifically expressed in primordia and fruiting bodies of the edible mushroom Pleurotus ostreatus. To understand its interaction with lipid membranes, we compared its effects on mammalian cells, on vesicles prepared with either pure lipids or total lipid extracts, and on dispersions of lysophospholipids or fatty acids. At nanomolar concentrations, the protein lysed human, bovine and sheep erythrocytes by a colloid-osmotic mechanism, compatible with the formation of pores of 4 nm diameter, and was cytotoxic to mammalian tumor cells. A search for lipid inhibitors of hemolysis revealed a strong effect of lysophospholipids and fatty acids, occurring below their critical micellar concentration. This effect was distinct from the capacity of ostreolysin to bind to and permeabilize lipid membranes. In fact, permeabilization of vesicles occurred only when they were prepared with lipids extracted from erythrocytes, and not with lipids extracted from P. ostreatus or pure lipid mixtures, even if lysophospholipids or fatty acids were included. Interaction with lipid vesicles, and their permeabilization, correlated with an increase in the intrinsic fluorescence and ,-helical content of the protein, and with aggregation, which were not detected with lysophospholipids. It appears that either an unknown lipid acceptor or a specific lipid complex is required for binding, aggregation and pore formation. The inhibitory effect of lysophospholipids may reflect a regulatory role for these components on the physiological action of ostreolysin and related proteins during fruiting. [source] Cytomorphological alterations of the thymus, spleen, head-kidney, and liver in cardinal fish (Apogonidae, Teleostei) as bioindicators of stressJOURNAL OF MORPHOLOGY, Issue 1 2006Lev Fishelson Abstract Morphological and cytological alterations at the light microscope (LM) and transmission electron microscope (TEM) levels were observed in the thymus, spleen, head-kidney, and liver of cardinal fishes (Apogonidae, Teleostei) from the Gulf of Aqaba, Red Sea, sampled from a strongly polluted site at the northern end of the gulf, and compared to similar samples from a clean, reference site. At the polluted site, the most prominent change was the formation of numerous deposits of cells rich in phagosomes with lipofucin, melanin granules, and phagocytosed debris, including a high increase in number and dimensions of Hassall's corpuscles and melano-macrophage centers. The number of Hassall's corpuscles was 20 (±8.0)/mm2 and of melano-macrophage centers 18 (±4.0)/mm2 at the polluted site, and 7.0 (±4.0)/m2 vs. 5.0 (±2.0)/mm2 respectively at the reference site. In numerous instances the head kidney's melano-macrophage centers in fishes from the polluted site were encapsulated by reticulocytes, a phenomenon recognized as a marker of neoplasmosis and possible malignancy. In the spleens of fishes from the polluted site, numerous deposits of cell debris, peroxisomes, and enlarged lysosomes were also observed. The livers (hepatopancreas) of fishes from polluted waters demonstrated very strong hyperlipogeny. Many of their hepatocytes were laden with lipid vesicles, fragmented endoplasmic reticulula, and aberrant mitochondria. Although the observed alterations in the glands and liver do not indicate any immediate threat to the life of the fish, they can become crucial with respect to energy turnover and fecundity trajectories. This study strongly suggests the use of cytological alterations in vital organs, such as were observed, as pathological biomarkers to environmental stress. J. Morphol. © 2005 Wiley-Liss, Inc. [source] Structure,activity relationship of an antibacterial peptide, maculatin 1.1, from the skin glands of the tree frog, Litoria genimaculataJOURNAL OF PEPTIDE SCIENCE, Issue 7 2004Takuro Niidome Abstract Maculatin 1.1 (Mac) is a cationic antibacterial peptide isolated from the dorsal glands of the tree frog, Litoria genimaculata, and has a sequence of GLFGVLAKVAAHVVPAIAEHF-NH2. A short peptide lacking the N -terminal two residues of Mac was reported to have no activity. To investigate the structure,activity relationship in detail, several analogs and related short peptides of Mac were synthesized. CD measurement showed that all the peptides took more or less an ,-helical structure in the presence of anionic lipid vesicles. Analogs which are more basic than Mac had strong antibacterial and hemolytic activities, while short peptides lacking one or two terminal residues exhibited weak or no activity. Outer and inner membrane permeabilization activities of the peptides were also reduced with shortening of the peptide chain. These results indicate that the entire chain length of Mac is necessary for full activity, and the basicity of the peptides greatly affects the activity. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd. [source] Parallel and antiparallel dimers of magainin 2: their interaction with phospholipid membrane and antibacterial activityJOURNAL OF PEPTIDE SCIENCE, Issue 10 2002Yasuhiro Mukai Abstract Magainin 2 (M2) forms pores by associating with several other M2 molecules in lipid membranes and shows antibacterial activity. To examine the effect of M2 dimerization on biological activity and membrane interaction, parallel and antiparallel M2 dimers were prepared from two monomeric precursors. Antibacterial and haemolytic activities were enhanced by dimerization. CD measurements showed that both dimers and monomers have an ,-helical structure in the presence of lipid vesicles. Tryptophan fluorescence shift and KI quenching studies showed that all the peptides were more deeply embedded in acidic liposomes than in neutral liposomes. Experiments on dye-leakage activity and membrane translocation of peptides suggest that dimers and monomers form pores through lipid membranes, although the pore formation may be accompanied by membrane disturbance. Although dimerization of M2 increased the interaction activity with lipid membranes, no appreciable difference between the activities of parallel and antiparallel M2 dimers was observed. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd. [source] Elastic liposomes mediated transdermal delivery of an anti-hypertensive agent: Propranolol hydrochlorideJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 1 2007Dinesh Mishra Abstract One major problem encountered in transdermal drug delivery is the low permeability of drugs through the skin barrier. In the present investigation ultradeformable lipid vesicles, that is, elastic liposomes were prepared incorporating propranolol hydrochloride for enhanced transdermal delivery. Elastic liposomes bearing propranolol hydrochloride were prepared by conventional rotary evaporation method and characterized for various parameters including vesicles shape and surface morphology, size and size distribution, entrapment efficiency, elasticity, turbidity, and in vitro drug release. In vitro flux, enhancement ratio (ER), and release pattern of propranolol hydrochloride were calculated for transdermal delivery. In vivo study conducted on male albino rats (Sprague Dawley) was also taken as a measure of performance of elastic liposomal, liposomal, and plain drug solution. The better permeation through the skin was confirmed by confocal laser scanning microscopy (CLSM). Results indicate that the elastic liposomal formulation for transdermal delivery of propranolol hydrochloride provides better transdermal flux, higher entrapment efficiency, ability as a self-penetration enhancer and effectiveness for transdermal delivery as compared to liposomes. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:145,155, 2007 [source] Intranasal immunization using biphasic lipid vesicles as delivery systems for OmlA bacterial protein antigen and CpG oligonucleotides adjuvant in a mouse modelJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 8 2005V. L. Alcón The nasal mucosa is an important arm of the mucosal system since it is often the first point of contact for inhaled antigens. The ineffectiveness of the simple delivery of soluble antigens to mucosal membranes for immunization has stimulated extensive studies in appropriate delivery systems and adjuvants. We have evaluated biphasic lipid vesicles as a novel intranasal (i.n.) delivery system (designated as vaccine targeting adjuvant, VTA) containing bacterial antigens and CpG oligode-oxynucleotides (ODNs). Results show that administration of antigen and CpG ODNs in biphasic lipid vesicles resulted in greater induction of IgA levels in serum (P<0.05) and mucosal antibody responses such as IgA in nasal secretions and lung (P<0.01) after immunization with a combined subcutaneous (s.c.)/i.n. as compared to s.c./s.c. approach. Based on antibody responses, VTA formulations were found to be suitable as delivery systems for antigens and CpG ODNs by the intranasal route, resulting in a Th2-type of immune response, characterized by IgG1 and IL-4 production at the systemic level. [source] Tetanus toxoid-loaded transfersomes for topical immunizationJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 3 2005Prem N. Gupta Topical immunization is a novel immunization strategy by which antigens and adjuvants are applied topically to intact skin to induce potent antibody and cell-mediated responses. Among various approaches for topical immunization, the vesicular approach is gaining wide attention. Proteineous antigen alone or in combination with conventional bioactive carriers could not penetrate through the intact skin. Hence, specially designed, deformable lipid vesicles called transfersomes were used in this study for the non-invasive delivery of tetanus toxoid (TT). Transfersomes were prepared and characterized for shape, size, entrapment efficiency and deformability index. Fluorescence microscopy was used to investigate the mechanism of vesicle penetration through the skin. The immune stimulating activity of these vesicles was studied by measuring the serum anti-tetanus toxoid IgG titre following topical immunization. The immune response was compared with the same dose of alum adsorbed tetanus toxoid (AATT) given intramuscularly, topically administered plain tetanus toxoid solution, and a physical mixture of tetanus toxoid and transfersomes again given topically. The results indicated that the optimal transfersomal formulation had a soya phosphatidylcholine and sodium deoxycholate ratio of 85:15%, w/w. This formulation showed maximum entrapment efficiency (87.34±3.81%) and deformability index (121.5±4.21). An in-vivo study revealed that topically administered tetanus toxoid-loaded transfersomes, after secondary immunization, elicited an immune response (anti-TT-IgG) comparable with that produced by intramuscular AATT. Fluorescence microscopy revealed the penetration of transfersomes through the skin to deliver the antigen to the immunocompetent Langerhans cells. [source] UV resonance Raman spectroscopy probes the localization of tryptophan-containing antimicrobial peptides in lipid vesiclesJOURNAL OF RAMAN SPECTROSCOPY, Issue 3 2009Bryan Quan Abstract In this work we employed UV resonance Raman spectroscopy with 229 nm excitation to study two tryptophan-containing antimicrobial peptides with a broad-spectrum activity against Gram-positive and Gram-negative bacteria: lactoferricin B (LfB, RRWQWRMKKLG) and pEM-2 (KKWRWWLKALAKK). UV resonance Raman spectra of both peptides are dominated by tryptophan bands. Raman spectra of LfB and pEM-2 in D2O and 2,2,2-trifluoro ethanol (TFE) have been measured and used to identify the hydrogen-bond strength marker bands W6 and W17. The tryptophan doublet, W7, at 1340 and 1360 cm,1 was used to detect an increase in the hydrophobicity of Trp environment in TFE. The spectra of LfB in complex with model cell membranes composed of zwitterionic dipalmitoylglycero-phosphocholine (DPPC) or anionic dipalmitoyglycero-phosphoglycerol (DPPG) lipid vesicles revealed a more hydrophobic Trp environment in DPPG, suggesting stronger interactions between the cationic peptide and anionic model cell membrane. Copyright © 2008 John Wiley & Sons, Ltd. [source] Orientation-Selective Incorporation of Transmembrane F0F1 ATP Synthase Complex from Micrococcus luteus in Polymer-Supported MembranesMACROMOLECULAR BIOSCIENCE, Issue 11 2008Murat Tutus Abstract We report the vectorial incorporation of a highly asymmetric F0F1 ATP synthase complex from Micrococcus luteus into polymer-supported membranes. Dynamic light scattering and cryo electron microscopy confirm that the use of weak surfactants (bile acid) allows for the non-disruptive protein incorporation into lipid vesicles. Spreading of vesicles with ATP synthase onto a cellulose support results in a homogeneous distribution of proteins, in contrast to a patchy image observed on bare glass slides. The orientation of ATP synthase can be identified using an antibody to the ATP binding site as well as from topographic profiles of the surface. The method to "align" transmembrane proteins in supported membranes would open a possibility to quantify protein functions in biomimetic model systems. [source] CFTR: More than just a chloride channelPEDIATRIC PULMONOLOGY, Issue 4 2005Anil Mehta MBBS, FRCP (Edin), FRCPCH Abstract This review examines the cystic fibrosis transmembrane conductance regulator (CFTR) protein. After summarizing the ion channels regulated by CFTR, the review focuses on the functions of CFTR that do not relate directly to a disease mechanism based on a channelopathy. The key concept is that newly synthesized CFTR has to enter lipid vesicles which bud from the endoplasmic reticulum. This is abnormally low in ,F508 CFTR. Normal wild type vesicular CFTR enters a recycling pool of lipid vesicles which transiently dock with the apical membrane only for CFTR to be retrieved shortly after into a sub-apical recycling compartment. This retrieval is abnormally fast in ,F508 CFTR. The review discusses the relationship between this process and the difficult topic of fat metabolism and then explores the possible links between abnormal fatty acid turnover and inflammatory cascades that are abnormal in cystic fibrosis. Finally the review concentrates on the emerging functions of a protein kinase (AMP-activated kinase) which is bound near the C terminus of the CFTR protein whose functions could intergrate some of the abnormalities in lipid metabolism that result from mislocalization of CFTR in clinical disease. Pediatr Pulmonol. 2005; 39:292,298. © 2004 Wiley-Liss, Inc. [source] High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptorsPROTEIN SCIENCE, Issue 9 2010Zuzana Dostalova Abstract The human neuronal Cys-loop ligand-gated ion channel superfamily of ion channels are important determinants of human behavior and the target of many drugs. It is essential for their structural characterization to achieve high-level expression in a functional state. The aim of this work was to establish stable mammalian cell lines that enable high-level heterologous production of pure receptors in a state that supports agonist-induced allosteric conformational changes. In a tetracycline-inducible stable human embryonic kidney cells (HEK293S) cell line, GABAA receptors containing ,1 and ,3 subunits could be expressed with specific activities of 29,34 pmol/mg corresponding to 140,170 pmol/plate, the highest expression level reported so far. Comparable figures for serotonin (5-HT3A) receptors were 49,63 pmol/mg and 245,315 pmol/plate. The expression of 10 nmol of either receptor in suspension in a bioreactor required 0.3,3.0 L. Both receptor constructs had a FLAG epitope inserted at the N-terminus and could be purified in one step after solubilization using ANTI-FLAG affinity chromatography with yields of 30,40%. Purified receptors were functional. Binding of the agonist [3H]muscimol to the purified GABAAR was enhanced allosterically by the general anesthetic etomidate, and purified 5-hydroxytryptamine-3A receptor supported serotonin-stimulated cation flux when reconstituted into lipid vesicles. [source] Liposome-bound APO2L/TRAIL is an effective treatment in a rabbit model of rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 8 2010Luis Martinez-Lostao Objective We previously observed that T lymphocytes present in synovial fluid (SF) from patients with rheumatoid arthritis (RA) were sensitive to APO2L/TRAIL. In addition, there was a drastic decrease in the amount of bioactive APO2L/TRAIL associated with exosomes in SF from RA patients. This study was undertaken to evaluate the effectiveness of bioactive APO2L/TRAIL conjugated with artificial lipid vesicles resembling natural exosomes as a treatment in a rabbit model of antigen-induced arthritis (AIA). Methods We used a novel Ni2+ -(N -5-amino-1-carboxypentyl)-iminodiacetic acid),containing liposomal system. APO2L/TRAIL bound to liposomes was intraarticularly injected into the knees of animals with AIA. One week after treatment, rabbits were killed, and arthritic synovial tissue was analyzed. Results Tethering APO2L/TRAIL to the liposome membrane increased its bioactivity and resulted in more effective treatment of AIA compared with soluble, unconjugated APO2L/TRAIL, with substantially reduced synovial hyperplasia and inflammation in rabbit knee joints. The results of biophysical studies suggested that the increased bioactivity of APO2L/TRAIL associated with liposomes was due to the increase in the local concentration of the recombinant protein, augmenting its receptor crosslinking potential, and not to conformational changes in the protein. In spite of this increase in bioactivity, the treatment lacked systemic toxicity and was not hepatotoxic. Conclusion Our findings indicate that binding APO2L/TRAIL to the liposome membrane increases its bioactivity and results in effective treatment of AIA. [source] Building artificial cells and protocell models: Experimental approaches with lipid vesiclesBIOESSAYS, Issue 4 2010Peter Walde Abstract Lipid vesicles are often used as compartment structures for preparing cell-like systems and models of protocells, the hypothetical precursor structures of the first cells at the origin of life. Although the various artificially made vesicle systems are already remarkably complex, they are still very different from and much simpler than any known living cell. Nevertheless, the preparation and study of the structure and the dynamics of functionalized vesicle systems may contribute to a better understanding of biological cells, in particular of the essential features of a living cell that are not found in the non-living form of matter. The study of protocell models may possibly lead to a better understanding of the origin of the first cells. To avoid misunderstanding in this field of research, it would be useful if generally accepted definitions of terms like "artificial cells," "synthetic cells," "minimal cells," "protocells," and "primitive cells" exist. , Editor's suggested further reading in BioEssays Synthetic cells and organelles: compartmentalization strategiesAbstract [source] The effect of temperature and lipid on the conformational transition of gramicidin A in lipid vesicles,BIOPOLYMERS, Issue 4 2005Ta-Hsien Lin Abstract The present study investigated the effect of temperature and lipid/peptide molar ratio on the conformational changes of the membrane peptide gramicidin A from a double-stranded helix to a single-stranded helical dimmer in 1,2-dimyristoyl-glycerol-3-phosphochloine (DMPC) vesicles. Tryptophan fluorescence spectroscopy results suggested that the conformational transition fitted a three-state (two-step) "folding" model. Rate constants, k1 and k2, were determined for each of the two steps. Since k1 and k2 increased with an increase in temperature, we hypothesized that the process corresponded to the breakage and formation of the backbone hydrogen bonds. The k1 was from 10 to 45 folds faster than k2, except for lipid/peptide molar ratios above 89.21, where k2 increased rapidly. At molar ratios below 89.21, k2 was insensitive to changes in lipid concentration. To account for this phenomenon, we proposed that while the driving interaction at high molar ratios is between the indole rings of the tryptophan residues and the lipid head groups, at low molar ratios there may be an intermolecular interaction between the tryptophan residues that causes gramicidin A to form an organized aggregated network. This aggregated network, caused by the tryptophan,tryptophan interaction, may be the main effect responsible for the slow down of the conformation change. © 2005 Wiley Periodicals, Inc. Biopolymers 78: 179,186, 2005 [source] Ortho-aminobenzoic acid-labeled bradykinins in interaction with lipid vesicles: Fluorescence studyBIOPOLYMERS, Issue 5 2002R. F. Turchiello Abstract The peptide hormone bradykinin (BK) (Arg1 -Pro2 -Pro3 -Gly4 -Phe5 -Ser6 -Pro7 -Phe8 -Arg9) and its shorter homolog BK1,5 (Arg1 -Pro2 -Pro3 -Gly4 -Phe5) were labeled with the extrinsic fluorescent probe ortho -aminobenzoic acid (Abz) bound to the N-terminal and amidated in the C-terminal carboxyl group (Abz-BK-NH2 and Abz-BK1,5 -NH2). The fragment des-Arg9 -BK was synthesized with the Abz fluorescent probe attached to the 3-amino group of 2,3-amino propionic acid (DAP), which positioned the Abz group at the C-terminal side of BK sequence, constituting the peptide des-Arg9 -BK-DAP(Abz)-NH2. The spectral characteristics of the probe were similar in the three peptides, and their fluorescent properties were monitored to study the interaction of the peptides with anionic vesicles of dimyristoylphosphatidylglycerol (DMPG). Time-resolved fluorescence experiments showed that the fluorescence decay of the peptides was best described by double-exponential kinetics, with mean lifetimes values around 8.0 ns in buffer pH 7.4 that increased about 10% in the presence of DMPG vesicles. About a 10-fold increase, compared with the values in aqueous solution, was observed in the steady-state anisotropy in the presence of vesicles. A similar increase was also observed for the rotational correlation times obtained from time-resolved anisotropy decay profiles, and related to the overall tumbling of the peptides. Equilibrium binding constants for the peptide,lipid interaction were examined monitoring anisotropy values in titration experiments and the electrostatic effects were evaluated through Gouy,Chapman potential calculations. Without corrections for electrostatic effects, the labeled fragment Abz-BK1,5 -NH2 presented the major affinity for DMPG vesicles. Corrections for the changes in peptide concentration due to electrostatic interactions suggested higher affinity of the BK fragments to the hydrophobic phase of the bilayer. © 2002 Wiley Periodicals, Inc. Biopolymers 65: 336,346, 2002 [source] Surface plasmon resonance for high-throughput ligand screening of membrane-bound proteinsBIOTECHNOLOGY JOURNAL, Issue 11 2009Jennifer A. Maynard Dr. Abstract Technologies based on surface plasmon resonance (SPR) have allowed rapid, label-free characterization of protein-protein and protein-small molecule interactions. SPR has become the gold standard in industrial and academic settings, in which the interaction between a pair of soluble binding partners is characterized in detail or a library of molecules is screened for binding against a single soluble protein. In spite of these successes, SPR is only beginning to be adapted to the needs of membrane-bound proteins which are difficult to study in situ but represent promising targets for drug and biomarker development. Existing technologies, such as BIAcoreTM, have been adapted for membrane protein analysis by building supported lipid layers or capturing lipid vesicles on existing chips. Newer technologies, still in development, will allow membrane proteins to be presented in native or near-native formats. These include SPR nanopore arrays, in which lipid bilayers containing membrane proteins stably span small pores that are addressable from both sides of the bilayer. Here, we discuss current SPR instrumentation and the potential for SPR nanopore arrays to enable quantitative, high-throughput screening of G protein coupled receptor ligands and applications in basic cellular biology. [source] Affinity Purification of Lipid VesiclesBIOTECHNOLOGY PROGRESS, Issue 1 2004Boris Peker We present a novel column chromatography technique for recovery and purification of lipid vesicles, which can be extended to other macromolecular assemblies. This technique is based on reversible binding of biotinylated lipids to monomeric avidin. Unlike the very strong binding of biotin and biotin-functionalized molecules to streptavidin, the interaction between biotin-functionalized molecules and monomeric avidin can be disrupted effectively by ligand competition from free biotin. In this work, biotin-functionalized lipids (biotin-PEG-PE) were incorporated into synthetic lipid vesicles (DOPC), resulting in unilamellar biotinylated lipid vesicles. The vesicles were bound to immobilized monomeric avidin, washed extensively with buffer, and eluted with a buffer supplemented with free biotin. Increasing the biotinyl lipid molar ratio beyond 0.53% of all lipids did not increase the efficiency of vesicle recovery. A simple adsorption model suggests 1.1 × 1013 active binding sites/mL of resin with an equilibrium binding constant of K = 1.0 × 108 M,1. We also show that this method is very robust and reproducible and can accommodate vesicles of varying sizes with diverse contents. This method can be scaled up to larger columns and/or high throughput analysis, such as a 96-well plate format. [source] Spontaneous Protein Crowding in Liposomes: A New Vista for the Origin of Cellular MetabolismCHEMBIOCHEM, Issue 14 2010Prof. Pier Luigi Luisi Ferritin encapsulation inside lipid vesicles reveals the spontaneous formation of protein-rich vesicles. The solute distribution inside the vesicles follows a power law. The important conclusion for origins-of-life scenarios is that the dynamics of membrane closure allow the accumulation of solutes inside primitive cells, thus providing an explanation for the origins of early functional cells. [source] Structure, biological activity and membrane partitioning of analogs of the isoprenylated a -factor mating peptide of Saccharomyces cerevisiaeCHEMICAL BIOLOGY & DRUG DESIGN, Issue 5 2000H. Xie Abstract: Previous biochemical investigations on the Saccharomyces cerevisiaea -factor indicated that this lipopeptide pheromone [YIIKGVFWDPAC(farnesyl)OMe] might adopt a type II ,-turn at positions 4 and 5 of the peptide sequence. To test this hypothesis, we synthesized five analogs of a -factor, in which residues at positions 4 and 5 were replaced with: l -Pro4(I); d -Pro4(II); l -Pro4 - d -Ala5(III); d - Pro4 - l -Ala5(IV); or Nle4(V). Analogs were purified to > 99% homogeneity as evidenced by HPLC and TLC and were characterized by mass spectrometry and amino acid analysis. Using a growth arrest assay the conformationally restricted a -factor analogs I and III were found to be almost 50-fold more active than the diastereometric homologs II and IV and were equally active to wild-type a -factor. Replacement of Lys4 with the isosteric Nle4 almost abolished the activity of the pheromone. Thus, the incorporation of residues that promote a type II ,-turn compensated for the loss of the favorable contribution of the Lys4 side chain to pheromone activity. CD spectra on these peptides suggested that they were essentially disordered in both TFE/H2O and in the presence of DMPC vesicles. There was no correlation between CD peak shape and biological activity. Using fluorescence spectroscopy we measured the interaction of lipid vesicles with these position 4 and 5 analogs as well as with three a -factor analogs with a modified farnesyl group. The results indicated that modifications of both the peptide sequence and the lipid moiety affect partitioning into lipid, and that no correlation existed between the propensity of a pheromone to partition into the lipid and its biological activity. [source] Surface-Functionalized Ultrasmall Superparamagnetic Nanoparticles as Magnetic Delivery Vectors for CamptothecinCHEMMEDCHEM, Issue 6 2009Feride Cengelli Abstract Drug,nanoparticle conjugates: The anticancer drug camptothecin (CPT) was covalently linked at the surface of ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) via a linker, allowing drug release by cellular esterases. Nanoparticles were hierarchically built to achieve magnetically-enhanced drug delivery to human cancer cells and antiproliferative activity. The linking of therapeutic drugs to ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs) allowing intracellular release of the active drug via cell-specific mechanisms would achieve tumor-selective magnetically-enhanced drug delivery. To validate this concept, we covalently attached the anticancer drug camptothecin (CPT) to biocompatible USPIOs (iron oxide core, 9,10,nm; hydrodynamic diameter, 52,nm) coated with polyvinylalcohol/polyvinylamine (PVA/aminoPVA). A bifunctional, end-differentiated dicarboxylic acid linker allowed the attachment of CPT to the aminoPVA as a biologically labile ester substrate for cellular esterases at one end, and as an amide at the other end. These CPT,USPIO conjugates exhibited antiproliferative activity in,vitro against human melanoma cells. The intracellular localization of CPT,USPIOs was confirmed by transmission electron microscopy (iron oxide core), suggesting localization in lipid vesicles, and by fluorescence microscopy (CPT). An external static magnetic field applied during exposure increased melanoma cell uptake of the CPT,USPIOs. [source] A Novel Heavy-Atom Label for Side-Specific Peptide Iodination: Synthesis, Membrane Incorporation and X-ray ReflectivityCHEMPHYSCHEM, Issue 9-10 2009Philipp E. Schneggenburger Abstract A novel iodine peptide label for X-ray analysis of membrane-active peptide structures is applied to solid-phase peptide synthesis. The resulting pore-structured labeled peptide as well as a non-labeled reference were reconstituted in lipid bilayer stacks (see scheme). The results indicate the exhibition of a membrane-spanning ,5.6 -double helical peptide structure and illustrate the quality of the new label. Structural parameters, such as conformation, orientation and penetration depth of membrane-bound peptides and proteins that may function as channels, pores or biocatalysts, are of persistent interest and have to be probed in the native fluid state of a membrane. X-ray scattering in combination with heavy-atom labeling is a powerful and highly appropriate method to reveal the position of a certain amino acid residue within a lipid bilayer with respect to the membrane normal axis up to a resolution of several Ångstrøm. Herein, we report the synthesis of a new iodine-labeled amino acid building block. This building block is intended for peptide incorporation to provide high intensities for electron density difference analysis of X-ray reflectivity data and improve the labeling potential for the lipid bilayer head-group and water region. The novel building block as well as the commercially available non-iodinated analogue, required for X-ray scattering, was implemented in a transmembrane peptide motif via manual solid-phase peptide synthesis (SPPS) following the fluorenylmethyloxycarbonyl (Fmoc)-strategy. The derived peptides were reconstituted in lipid vesicles as well as in highly aligned multilamellar lipid stacks and investigated via circular dichroism (CD) and X-ray reflectivity. Thereby, it has been revealed that the bulky iodine probe neither causes conformational change of the peptide structure nor lamellar disordering of the membrane complexes. [source] | ||||||||||||||||