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201.
Stabilized plasmid lipid particles (SPLP) consist of a single copy of DNA surrounded by a lipid bilayer. The particles are small (∼100 nm), stable, monodisperse and have a low surface charge. A diffusible polyethylene glycol (PEG) coating attached to a lipid anchor is critical to the SPLP's functionality. The PEG-lipid exchanges out of the bilayer at a rate determined by the size of the lipid anchor. Here we show that SPLP can be prepared using a series of PEG-diacylglycerol lipids (PEG-S-DAGs). SPLP were prepared incorporating PEG-dimyristoylglycerol (C14), PEG-dipalmitoylglycerol (C16) or PEG-distearoylglycerol (C18) and the rate of PEG-lipid diffusion from the bi-layer determined using a FRET assay. SPLP pharmacokinetics confirm a correlation between the stability of the PEG-lipid component and circulation lifetime. PEG-S-DAGs with longer lipid anchors yield more stable SPLP particles with longer circulation half-lives yielding an increase in tumor delivery and gene expression. PEG-distearoylglycerol (C18) containing SPLP bypass so-called ‘first pass’ organs, including the lung, and elicit levels of gene expression in distal tumor tissue 100- to 1000-fold greater than that observed in any other tissue. The incorporation of PEG-S-DAG in SPLP confirms that small size, low surface charge and extended circulation lifetimes are prerequisite to the accumulation and tumor selective expression of plasmid DNA following systemic administration. 相似文献
202.
A new and unnatural type of lipid analogs with the phosphocholine and phosphoglycerol head groups linked to the C-2 position of the glycerol moiety have been synthesized and the thermodynamic lipid membrane behavior has been investigated using differential scanning calorimetry. From the heat capacity measurements, it was observed that the pre-transition was abolished most likely due to the central position of the head groups providing better packing properties in the low temperature ordered gel phase. Activity measurements of secretory phospholipase A2 (PLA2) on unilamellar liposomal membranes revealed that the unnatural phospholipids are excellent substrates for PLA2 catalyzed hydrolysis. This was manifested as a minimum in the PLA2 lag time in the main phase transition temperature regime and a high degree of lipid hydrolysis over a broad temperature range. The obtained results provide new information about the interplay between the molecular structure of phospholipids and the lipid membrane packing constrains that govern the pre-transition. In addition, the PLA2 activity measurements are useful for obtaining deeper insight into the molecular details of the catalytic site of PLA2. The combined results also suggest new approaches to rationally design liposomal drug carries that can undergo a triggered activation in diseased tissue by overexpressed PLA2. 相似文献
203.
Stimulated echo pulsed-field gradient 1H magic angle spinning NMR has been used to investigate the mobility of water, ubiquinone and tethered phospholipids, components of a biomimetic model membrane. The diffusion constant of water corresponds to an isotropic motion in a cylinder. When the lipid bilayer is obtained after the fusion of small unilamellar vesicles, the extracted value of lipid diffusion indicates unrestricted motion. The cylindrical arrangement of the lipids permits a simplification of data analysis since the normal bilayer is perpendicular to the gradient axis. This feature leads to a linear relation between the logarithm of the attenuation of the signal intensity and a factor depending on the gradient strength, for lipids covering the inner wall of aluminium oxide nanopores as well as for lipids adsorbed on a polymer sheet rolled into a cylinder. The effect of the bilayer formation on water diffusion has also been observed. The lateral diffusion coefficient of ubiquinone is in the same order of magnitude as the lipid lateral diffusion coefficient, in agreement with its localization within the bilayer. 相似文献
204.
Yukiko Matsuo Tsutomu Ishihara Ken-ichi Miyamoto Naomi Yamashita 《Cellular immunology》2009,260(1):33-38
Although inhaled steroids are the treatment of first choice to control asthma, administration of systemic steroids is required for treatment of asthmatic exacerbation and intractable asthma. To improve efficacy and reduce side effects, we examine the effects of betamethasone disodium phosphate (BP) encapsulated in biocompatible, biodegradable blended nanoparticles (stealth nanosteroids) on a murine model of asthma. These stealth nanosteroids were found to accumulate at the site of airway inflammation and exhibit anti-inflammatory activity. Significant decreases in BALF eosinophil number were maintained for 7 days with a single injection of nanosteroids containing 40 μg BP. Airway responsiveness was also attenuated by the injection of stealth nanosteroids. A single injection of 40 μg of free BP and 8 μg of free BP once daily for 5 days did not show any significant effects. We conclude that stealth nanosteroids achieve prolonged and higher benefits at the site of airway inflammation compared to free steroids. 相似文献
205.
Although aerobic degradation of ethylene glycol is well documented, only anaerobic biodegradation via methanogenesis or fermentation
has been clearly shown. Enhanced ethylene glycol degradation has been demonstrated by microorganisms in the rhizosphere of
shallow-rooted plants such as alfalfa and grasses where conditions may be aerobic, but has not been demonstrated in the deeper
rhizosphere of poplar or willow trees where conditions are more likely to be anaerobic. This study evaluated ethylene glycol
degradation under nitrate-, and sulphate-reducing conditions by microorganisms from the rhizosphere of poplar and willow trees
planted in the path of a groundwater plume containing up to 1.9 mol l−1 (120 g l−1) ethylene glycol and, the effect of fertilizer addition when nitrate or sulphate was provided as a terminal electron acceptor
(TEA). Microorganisms in these rhizosphere soils degraded ethylene glycol using nitrate or sulphate as TEAs at close to the
theoretical stoichiometric amounts required for mineralization. Although the added nitrate or sulphate was primarily used
as TEA, TEAs naturally present in the soil or CO2 produced from ethylene glycol degradation were also used, demonstrating multiple TEA usage. Anaerobic degradation produced
acetaldehyde, less acetic acid, and more ethanol than under aerobic conditions. Although aerobic degradation rates were faster,
close to 100% disappearance was eventually achieved anaerobically. Degradation rates under nitrate-reducing conditions were
enhanced upon fertilizer addition to achieve rates similar to aerobic degradation with up to 19.3 mmol (1.20 g) of ethylene
glycol degradation l−1 day−1 in poplar soils. This is the first study to demonstrate that microorganisms in the rhizosphere of deep rooted trees like
willow and poplar can anaerobically degrade ethylene glycol. Since anaerobic biodegradation may significantly contribute to
the phytoremediation of ethylene glycol in the deeper subsurface, the need for “pump and treat” or an aerobic treatment would
be eliminated, hence reducing the cost of treatment. 相似文献
206.
Sandro Carrara Luca Benini Vijayender Bhalla Claudio Stagni Anna Ferretti Andrea Cavallini Bruno Ricc Bruno Samorì 《Biosensors & bioelectronics》2009,24(12):3425-3429
This paper examines reliable advancements in low-cost DNA- and immuno-chips. Capacitance detection was successfully chosen to develop label-free bio-chips. Probe immobilization was rigorously investigated in order to obtain reliable capacitance measurements. Protein probes immobilized by using usual alkanethiols or thiolated ssDNA probes directly immobilized on gold do not allow sufficient stable capacitance measurements. New alkanethiols improved with ethylene–glycol function are shown in this paper to be more suitable materials for capacitive bio-chip development. Atomic Force Microscopy, Quartz Crystal Microbalance, and Capacitance Measurements were used to demonstrate that ethylene–glycol alkanethiols allow high time stability, smaller errors in detection, and improved ideal behaviour of the sensing surfaces. Measured capacitance is in the range of 8–11 nF/mm2 for antibody layers and close to 6 nF/mm2 for DNA probes. It is in the range of 10–12 nF/mm2 and of 4–6 nF/mm2 for antigen and DNA detection, respectively. The percentage error in detection is highly improved and it is in the range of 11–37% and of 0,23–0,82% for antigen and DNA, respectively. The reproducibility is also improved and it is close to 0,44% for single spot measurements on ethylene–glycol alkanethiols. A molecular theory attributing these improvements to water molecules strongly coordinated by ethylene–glycol functional groups and to solution ions not entering into probe films is finally proposed. 相似文献
207.
The addition of various polymers to pulmonary surfactants improves surface activity in experiments both in vitro and in vivo. Although the viscosity of surfactants has been investigated, the viscosity of surfactant polymer mixtures has not. In this study, we have measured the viscosities of Survanta and Infasurf with and without the addition of polyethylene glycol, dextran or hyaluronan. The measurements were carried out over a range of surfactant concentrations using two concentrations of polymers at two temperatures. Our results indicate that at lower surfactant concentrations, the addition of any polymers increased the viscosity. However, the addition of polyethylene glycol and dextran to surfactants at clinically used concentrations can substantially lower viscosity. Addition of hyaluronan at clinical surfactant concentrations slightly increased Infasurf viscosity and produced little change in Survanta viscosity. Effects of polymers on viscosity correlate with changes in size and distribution of surfactant aggregates and the apparent free volume of liquid as estimated by light microscopy. Aggregation of surfactant vesicles caused by polymers may therefore not only improve surface activity as previously shown, but may also affect viscosity in ways that could improve surfactant distribution in vivo. 相似文献
208.
We report a high-throughput phage selection method to identify mutants of Sfp phosphopantetheinyl transferase with altered substrate specificities from a large library of the Sfp enzyme. In this method, Sfp and its peptide substrates are co-displayed on the M13 phage surface as fusions to the phage capsid protein pIII. Phage-displayed Sfp mutants that are active with biotin-conjugated coenzyme A (CoA) analogues would covalently transfer biotin to the peptide substrates anchored on the same phage particle. Affinity selection for biotin-labeled phages would enrich Sfp mutants that recognize CoA analogues for carrier protein modification. We used this method to successfully change the substrate specificity of Sfp and identified mutant enzymes with more than 300-fold increase in catalytic efficiency with 3′-dephospho CoA as the substrate. The method we developed in this study provides a useful platform to display enzymes and their peptide substrates on the phage surface and directly couples phage selection with enzyme catalysis. We envision this method to be applied to engineering the catalytic activities of other protein posttranslational modification enzymes. 相似文献
209.
Structural and Functional Analysis of SoPIP2;1 Mutants Adds Insight into Plant Aquaporin Gating 总被引:1,自引:0,他引:1
Maria Nyblom Yi Wang Karin Hallgren Richard Neutze Susanna Törnroth-Horsefield 《Journal of molecular biology》2009,387(3):653-1335
Plant plasma membrane aquaporins facilitate water flux into and out of plant cells, thus coupling their cellular function to basic aspects of plant physiology. Posttranslational modifications of conserved phosphorylation sites, changes in cytoplasmic pH and the binding of Ca2+ can regulate water transport activity by gating the plasma membrane aquaporins. A structural mechanism unifying these diverse biochemical signals has emerged for the spinach aquaporin SoPIP2;1, although several questions concerning the opening mechanism remain. Here, we describe the X-ray structures of the S115E and S274E single SoPIP2;1 mutants and the corresponding double mutant. Phosphorylation of these serines is believed to increase water transport activity of SoPIP2;1 by opening the channel. However, all mutants crystallised in a closed conformation, as confirmed by water transport assays, implying that neither substitution fully mimics the phosphorylated state. Nevertheless, a half-turn extension of transmembrane helix 1 occurs upon the substitution of Ser115, which draws the Cα atom of Glu31 10 Å away from its wild-type conformation, thereby disrupting the divalent cation binding site involved in the gating mechanism. Mutation of Ser274 disorders the C-terminus but no other significant conformational changes are observed. Inspection of the hydrogen-bond interactions within loop D suggested that the phosphorylation of Ser188 may also produce an open channel, and this was supported by an increased water transport activity for the S188E mutant and molecular dynamics simulations. These findings add additional insight into the general mechanism of plant aquaporin gating. 相似文献
210.
The Ca2+-independent membrane interactions of the soluble C2 domains from synaptotagmin 1 (syt1) were characterized using a combination of site-directed spin labeling and vesicle sedimentation. The second C2 domain of syt1, C2B, binds to membranes containing phosphatidylserine and phosphatidylcholine in a Ca2+-independent manner with a lipid partition coefficient of approximately 3.0 × 102 M− 1. A soluble fragment containing the first and second C2 domains of syt1, C2A and C2B, has a similar affinity, but C2A alone has no detectable affinity to phosphatidylcholine/phosphatidylserine bilayers in the absence of Ca2+. Although the Ca2+-independent membrane affinity of C2B is modest, it indicates that this domain will never be free in solution within the cell. Site-directed spin labeling was used to obtain bilayer depth restraints, and a simulated annealing routine was used to generate a model for the membrane docking of C2B in the absence of Ca2+. In this model, the polybasic strand of C2B forms the membrane binding surface for the domain; however, this face of C2B does not penetrate the bilayer but is localized within the aqueous double layer when C2B is bound. This double-layer location indicates that C2B interacts in a purely electrostatic manner with the bilayer interface. In the presence of Ca2+, the membrane affinity of C2B is increased approximately 20-fold, and the domain rotates so that the Ca2+-binding loops of C2B insert into the bilayer. This Ca2+-triggered conformational change may act as a switch to modulate the accessibility of the polybasic face of C2B and control interactions of syt1 with other components of the fusion machinery. 相似文献