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51.
Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters <100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter <100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes <100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below. 相似文献
52.
Bakker BM Overkamp KM van Maris AJ Kötter P Luttik MA van Dijken JP Pronk JT 《FEMS microbiology reviews》2001,25(1):15-37
In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments. 相似文献
53.
54.
Castagnone-Sereno P; Semblat JP; Leroy F; Abad P 《Molecular biology and evolution》1998,15(9):1115-1122
A highly abundant satellite DNA comprising 20% of the Meloidogyne fallax
(Nematoda, Tylenchida) genome was cloned and sequenced. The satellite
monomer is 173 bp long and has a high A + T content of 72.3%, with frequent
runs of A's and T's. The sequence variability of the monomers is 2.7%,
mainly due to random distribution of single-point mutations. A search for
evidence of internal repeated subunits in the monomer sequence revealed a
6-bp motif (AAATTT) for which five degenerated repeats, differing by just a
single base pair, could be identified. Pairwise comparison of the M. fallax
satellite with those from the sympatric species Meloidogyne chitwoodi and
Meloidogyne hapla revealed a high sequence similarity (68.39%) with one
satellite DNA subfamily in M. chitwoodi, which indicated an unexpected
close relationship between them. Given the high copy number and the extreme
sequence homogeneity among monomeric units, it may be assumed that the
satellite DNA of M. fallax could have evolved through some recent and
extensive amplification burst in the nematode genome. In this case, its
relatively short life would not yet have allowed the accumulation of random
mutations in independent amplified repeats. Considering the morphological
resemblance between the two species and their ability to produce
interspecific fertile hybrids under controlled conditions, these results
indicate that M. fallax may share a common ancestor with M. chitwoodi, from
which it could have diverged recently. All these data suggest that M.
fallax could be the result of a recent speciation process and show that
Meloidogyne satellite DNAs may be of interest to resolve phylogenetic
relationships among closely related species from this genus.
相似文献
55.
56.
Vincent Ranwez Frédéric Delsuc Sylvie Ranwez Khalid Belkhir Marie-Ka Tilak Emmanuel JP Douzery 《BMC evolutionary biology》2007,7(1):241
Background
Molecular sequence data have become the standard in modern day phylogenetics. In particular, several long-standing questions of mammalian evolutionary history have been recently resolved thanks to the use of molecular characters. Yet, most studies have focused on only a handful of standard markers. The availability of an ever increasing number of whole genome sequences is a golden mine for modern systematics. Genomic data now provide the opportunity to select new markers that are potentially relevant for further resolving branches of the mammalian phylogenetic tree at various taxonomic levels. 相似文献57.
58.
59.
Solveig Tangedal Marianne Aanerud Louise JP Persson Karl A Brokstad Per S Bakke Tomas M Eagan 《Respiratory research》2014,15(1)
Background
Sputum induction is a non-invasive method for obtaining measurements of inflammation in the airways. Whether spontaneously sampled sputum can be a valid surrogate is unknown. The aim of this study was to compare levels of six inflammatory markers in sputum pairs consisting of induced and spontaneous sputum sampled on the same consultation either in a stable state or during exacerbations of chronic obstructive pulmonary disease (COPD).Methods
433 COPD patients aged 40–76, Global initiative for chronic Obstructive Lung Disease (GOLD) stage II-IV were enrolled in 2006/07 and followed every six months for three years. 356 patients were followed for potential exacerbations. Interleukin-6, interleukin-8, interleukin-18, interferon gamma-inducible protein-10, monokine induced by gamma interferon and tumor necrosis factor-alpha (IL-6, IL-8, IL-18, IP-10, MIG and TNF-α) were measured by bead based multiplex immunoassay in 60 paired sputum samples from 45 patients. Albumin was measured by enzyme immunoassay, for concentration correction. Culturing for bacterial growth was performed on 24 samples. Bland-Altman plots were used to assess agreement. The paired non-parametric Wilcoxon signed-rank test, the non-parametric Spearman’s rank correlation test and Kruskal-Wallis test were used for statistical analyses. For all analyses, a p-value < 0.05 was considered significant.Results
Agreement between the two measurements was generally low for all six markers. TNF-α was significantly higher in spontaneous sputum at exacerbations (p = 0.002) and trending higher at the steady state (p = 0.06). Correlation coefficients between the levels of markers in induced and spontaneous sputum varied between 0.58 (IL-18) to 0.83 (IP-10). In spontaneous sputum IL-18 and MIG were higher in ex-smokers (p < 0.05). The levels of all markers were higher in GOLD stage III & IV except for IL-6 in spontaneous sputum and IL-18 in induced sputum, compared with GOLD stage II, although not statistically significant. In spontaneous sputum the levels of IL-6 were significantly higher if Haemophilus influenzae (HI) was not cultured.Conclusion
We observed a low agreement and significant differences in inflammatory markers between induced and spontaneous sputum, both at steady state and exacerbations. We recommend considering sampling method when reporting on inflammatory markers in sputum. 相似文献60.