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1.
The release of granulysin, a 9-kDa cationic protein, from lysosomal granules of cytotoxic T lymphocytes and natural killer cells plays an important role in host defense against microbial pathogens. Granulysin is endocytosed by the infected target cell via lipid rafts and kills subsequently intracellular bacteria. The mechanism by which granulysin binds to eukaryotic and prokaryotic cells but lyses only the latter is not well understood. We have studied the effect of granulysin on large unilamellar vesicles (LUVs) and supported bilayers with prokaryotic and eukaryotic lipid mixtures or model membranes with various lipid compositions and charges. Binding of granulysin to bilayers with negative charges, as typically found in bacteria and lipid rafts of eukaryotic cells, was shown by immunoblotting. Fluorescence release assays using LUV revealed an increase in permeability of prokaryotic, negatively charged and lipid raft-like bilayers devoid of cholesterol. Changes in permeability of these bilayers could be correlated to defects of various sizes penetrating supported bilayers as shown by atomic force microscopy. Based on these results, we conclude that granulysin causes defects in negatively charged cholesterol-free membranes, a membrane composition typically found in bacteria. In contrast, granulysin is able to bind to lipid rafts in eukaryotic cell membranes, where it is taken up by the endocytotic pathway, leaving the cell intact.  相似文献   

2.
Lipids are the principal components of biologically relevant structures as cellular membranes. They have been the subject of many studies due to their biological relevance and their potential applications. Different techniques, such as Langmuir-Blodgett and vesicle-fusion deposition, are available to deposit ordered lipid films on etched surfaces. Recently, a new technique of lipid film deposition has been proposed in which stacks of a small and well-controlled number of bilayers are prepared on a suitable substrate using a spin-coater.We studied the morphological properties of multi-layers made of cationic and neutral lipids (DOTAP and DOPC) and mixtures of them using dynamic mode atomic force microscopy (AFM). After adapting and optimizing, the spin-coating technique to deposit lipids on a chemically etched Silicon (1,0,0) substrate, a morphological nanometer-scale characterization of the aforementioned samples has been provided. The AFM study showed that an initial layer of ordered vesicles is formed and, afterward, depending on details of the spin-coating preparation protocol and to the dimension of the silicon substrate, vesicle fusion and structural rearrangements of the lipid layers may occur.The present data disclose the possibility to control the lipid's structures by acting on spin-coating parameters with promising perspectives for novel applications of lipid films.  相似文献   

3.
Understanding drug-biomembrane interactions at high resolution is a key issue in current biophysical and pharmaceutical research. Here we used real-time atomic force microscopy (AFM) imaging to visualize the interaction of the antibiotic azithromycin with lipid domains in model biomembranes. Various supported lipid bilayers were prepared by fusion of unilamellar vesicles on mica and imaged in buffer solution. Phase-separation was observed in the form of domains made of dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), or SM/cholesterol (SM/Chl) surrounded by a fluid matrix of dioleoylphosphatidylcholine (DOPC). Time-lapse images collected following addition of 1 mM azithromycin revealed progressive erosion and disappearance of DPPC gel domains within 60 min. We attribute this effect to the disruption of the tight molecular packing of the DPPC molecules by the drug, in agreement with earlier biophysical experiments. By contrast, SM and SM-Chl domains were not modified by azithromycin. We suggest that the higher membrane stability of SM-containing domains results from stronger intermolecular interactions between SM molecules. This work provides direct evidence that the perturbation of lipid domains by azithromycin strongly depends on the lipid nature and opens the door for developing new applications in membrane biophysics and pharmacology.  相似文献   

4.
An isocratic high-performance liquid chromatographic method with column switching and direct injection has been developed to determine ciprofloxacin in plasma and Mueller–Hinton broth. An on-line dilution of the sample was performed with a loading mobile phase consisting of 173 mM phosphoric acid. The analyte was retained on a LiChrocart 4-4 precolumn filled with a LiChrospher 100 RP18, 5 μm. An electric-actuated system with two six-port valves allowed a clean-up step with a mixture 20 mM phosphate buffer (pH 3.5)–methanol (97: 3, v/v) and the transfer of the analyte by a back-flush mode to a 150×4.6 mm I.D. column packed with a Kromasil C8 5 μm, using a mobile phase of 20 mM phosphate buffer (pH 3.5)–acetonitrile (85:15, v/v). Fluorescence detection allowed a quantification limit of 0.078 μg/ml with a 40-μl sample size. The method was evaluated to determine its usefulness in studying the pharmacokinetic/pharmacodynamic behaviour of ciprofloxacin in an in vitro model.  相似文献   

5.
There is substantial evidence which implicates α-synuclein and its ability to aggregate and bind vesicle membranes as critical factors in the development of Parkinson's disease. In order to investigate the interaction between α-synuclein wild type (Wt) and its familial mutants, A53T and A30P with lipid membranes, we developed a novel lipid binding assay using surface enhanced laser desorption/ionisation-time of flight-mass spectrometry (SELDI-TOF MS). Wt and A53T exhibited similar lipid binding profiles; monomeric species and dimers bound with high relative affinity to the lipid surface, the latter of which exhibited preferential binding. Wt and A53T trimers and tetramers were also detected on the lipid surface. A30P exhibited a unique lipid binding profile; monomeric A30P bound with a low relative affinity, however, the dimeric species of A30P exhibited a higher binding ability. Larger order A30P oligomers were not detected on the lipid surface. Tapping mode atomic force microscopy (AFM) imaging was conducted to further examine the α-synuclein-lipid interaction. AFM analysis revealed Wt and its familial mutants can penetrate lipid membranes or disrupt the lipid and bind the hydrophobic alkyl self-assembled monolayer (SAM) used to form the lipid layer. The profile of these studied proteins revealed the presence of ‘small features’ consistent with the presence of monomeric and dimeric forms of the protein. These data collectively indicate that the dimeric species of Wt and its mutants can bind and cause membrane perturbations.  相似文献   

6.
    
Insulin has long been served as a model for protein aggregation, both due to the importance of aggregation in the manufacture of insulin and because the structural biology of insulin has been extensively characterized. Despite intensive study, details about the initial triggers for aggregation have remained elusive at the molecular level. We show here that at acidic pH, the aggregation of insulin is likely initiated by a partially folded monomeric intermediate. High-resolution structures of the partially folded intermediate show that it is coarsely similar to the initial monomeric structure but differs in subtle details—the A chain helices on the receptor interface are more disordered and the B chain helix is displaced from the C-terminal A chain helix when compared to the stable monomer. The result of these movements is the creation of a hydrophobic cavity in the center of the protein that may serve as nucleation site for oligomer formation. Knowledge of this transition may aid in the engineering of insulin variants that retain the favorable pharamacokinetic properties of monomeric insulin but are more resistant to aggregation.  相似文献   

7.
Biomembranes are not homogeneous, they present a lateral segregation of lipids and proteins which leads to the formation of detergent-resistant domains, also called “rafts”. These rafts are particularly enriched in sphingolipids and cholesterol. Despite the huge body of literature on raft insolubility in non-ionic detergents, the mechanisms governing their resistance at the nanometer scale still remain poorly documented. Herein, we report a real-time atomic force microscopy (AFM) study of model lipid bilayers exposed to Triton X-100 (TX-100) at different concentrations. Different kinds of supported bilayers were prepared with dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM) and cholesterol (Chol). The DOPC/SM 1:1 (mol/mol) membrane served as the non-resistant control, and DOPC/SM/Chol 2:1:1 (mol/mol/mol) corresponded to the raft-mimicking composition. For all the lipid compositions tested, AFM imaging revealed that TX-100 immediately solubilized the DOPC fluid phase leaving resistant patches of membrane. For the DOPC/SM bilayers, the remaining SM-enriched patches were slowly perforated leaving crumbled features reminiscent of the initial domains. For the raft model mixture, no holes appeared in the remaining SM/Chol patches and some erosion occurred. This work provides new, nanoscale information on the biomembranes' resistance to the TX-100-mediated solubilization, and especially about the influence of Chol.  相似文献   

8.
The phytopathogen Pseudomonas syringae pv. syringae produces toxic cyclic lipodepsipeptides (CLPs): nona-peptides and syringopeptins. All CLPs inhibit the growth of many fungal species, including human pathogens, although different fungi display different degrees of sensitivity. The best studied CLPs are Syringomycin-E (SR-E), Syringotoxin-B (ST-B) and Syringopeptin-25A (SP-25A). Their biological activity is affected by membrane composition and their structural differences. We previously (Matyus et al. in Eur Biophys J 35:459–467, 2006) reported the molecular features and structural preferences of SR-E in water and octane environments. Here we investigate in atomic detail the molecular features of the two other main CLP components, ST-B and SP-25A, in water and octane by 200 ns molecular dynamics simulations (MD), using distance restraints derived from NMR NOE data (Ballio et al. in Eur J Biochem 234:747–758, 1995). We have obtained three-dimensional models of ST-B and SP-25A CLPs in different environments. These models can now be used as a basis to investigate the interactions of ST-B and SP-25A with lipid membranes an important further step towards a better understanding of the antifungal and antibacterial activity of these peptides. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

9.
We employ an implementation of rapid-scan Fourier transform infrared (FT-IR) microspectroscopic imaging to acquire time-resolved images for assessing the non-repetitive reorganizational dynamics of aqueous dispersions of multilamellar lipid vesicles (MLVs) derived from distearoylphosphatidylcholine (DSPC). The spatially and temporally resolved images allow direct and simultaneous determinations of various physical and chemical properties of the MLVs, including the main thermal gel to liquid crystalline phase transition, comparisons of vesicle diffusion rates in both phases and the variation in lipid bilayer packing properties between the inner and outer lamellae defining the vesicle. Specifically, in the lipid liquid crystalline phase, the inner bilayers of the MLVs are more intermolecularly ordered than the outer regions, while the intramolecular acyl chain order/disorder parameters, reflecting the overall characteristics of the fluid phase, remain uniform across the vesicle diameter. In contrast, the lipid vesicle gel phase displays no intermolecular or intramolecular dependence as a function of distance from the MLV center.  相似文献   

10.
荧光相关谱技术及其应用   总被引:2,自引:1,他引:2  
基于对处于平衡态少量荧光分子集合的强度涨落进行时间平均的技术,荧光相关谱fluoreswceance correlation spectroscopy,FCS)技术最近已经应用于细胞环境过程的研究。FCS优秀的灵敏特性为我们实时测量许多参数提供了途径,而且具有快速的时间特性和高空间分辨率。测量的参数包括扩散速率、局部浓度、聚合状态和分子间的相互作用。荧光互相关谱(fluorescence cross-correlation spectroscopy,FCCS)进一步扩展了FCS技术的应用,包括在活细胞中的广泛应用。本文介绍了FCS技术的原理、实验装置及其应用。  相似文献   

11.
A method to determine plasma concentrations of ciprofloxacin and its metabolite desethyleneciprofloxacin (M1) by CE with HeCd laser-induced fluorescence detection is described. Following precipitation of proteins and centrifugation supernatant is injected hydrodynamically (10 s, 0.5 p.s.i.) into the capillary. Overall analysis time for the quantification of both analytes was 7 min. The total amount of plasma needed for multiple injections (n>5) was 10–20 μl. Data on accuracy and precision are presented. The assay performance is compared to the specifications of a validated HPLC method, which is routinely used for the quantification of ciprofloxacin and M1 in body fluids. Both methods showed comparable accuracy and precision for both analytes throughout the whole working range (inter-day precision <9%; inter-day accuracy 96–110%). The limit of quantification (LOQ) of 20 μg/l (M1 10 μg/l) for the CE procedure was slightly higher than for the HPLC method, where 10 μg/l (M1 2.5 μg/l) was determined. However, application of the methods to human plasma samples derived from a clinical study proved that comparable results are obtained and that the sensivity of the HPCE method was sufficient to fully describe typical plasma concentration timie profiles of ciprofloxacin and its metabolite M1. Both the adequate sensitivity and the required smaller sample volume compared to HPLC indicate that the method is feasible for clinical studies where sample amounts are limited, e.g., studies to investigate pharmacokinetics in pediatric patients. Preclinical studies form another possible application of this technique.  相似文献   

12.
    
Conformational changes of proteins and other biomolecules play a fundamental role in their functional mechanism. Single pair Förster resonance energy transfer (spFRET) offers the possibility to detect these conformational changes and dynamics, and to characterize their underlying kinetics. Using spFRET on microscopes with different modes of detection, dynamic timescales ranging from nanoseconds to seconds can be quantified. Confocal microscopy can be used as a means to analyze dynamics in the range of nanoseconds to milliseconds, while total internal reflection fluorescence (TIRF) microscopy offers information about conformational changes on timescales of milliseconds to seconds. While the existence of dynamics can be directly inferred from the FRET efficiency time trace or the correlation of FRET efficiency and fluorescence lifetime, additional computational approaches are required to extract the kinetic rates of these dynamics, a short overview of which is given in this review.
  相似文献   

13.
Without any exaggeration, cholesterol is one of the most important lipid species in eukaryotic cells. Its effects on cellular membranes and functions range from purely mechanistic to complex metabolic ones, besides which it is also a precursor of the sex hormones (steroids) and several vitamins. In this review, we discuss the biophysical effects of cholesterol on the lipid bilayer, in particular the ordering and condensing effects, concentrating on the molecular level or inter-atomic interactions perspective, starting from two-component systems and proceeding to many-component ones e.g., modeling lipid rafts. Particular attention is paid to the roles of the methyl groups in the cholesterol ring system, and their possible biological function. Although our main research methodology is computer modeling, in this review we make extensive comparisons between experiments and different modeling approaches.  相似文献   

14.
The aim of the present study was to determine the effects of the bovine serum albumin (BSA) and fetal calf serum (FCS) on sperm quality, DNA fragmentation and lipid peroxidation of liquid stored rabbit semen stored up to 72 h at 5 °C. Ejaculates were collected from five New Zealand male rabbits by artificial vagina and pooled at 37 °C following evaluation. Each pooled ejaculate was split into three equal experimental groups and diluted to a final concentration of approximately 40 × 106 sperm/ml (single step dilution), in an Eppendorf tube, with the Tris based extender containing BSA (5 mg/ml), FCS (10%) or no additive (control) at 37 °C, cooled to 5 °C and stored for up to 72 h. The extender supplemented with BSA and FCS did not improve the percentages of motility and acrosomal abnormality during 48 h compared to the control. The additives BSA and FCS had a significant effect in the maintaining of plasma membrane integrity between 48 and 72 h storage period, compared to the control (P < 0.01). The supplementation of BSA and FCS had a protective effect on motility (P < 0.05), plasma membrane integrity (P < 0.01) and acrosomal integrity (P < 0.01) at 72 h compared to the control. The supplementations with BSA and FCS led to a reduction in DNA damage of rabbit sperm at 48 and 72 h during storage period, compared to the control (P < 0.001). Although supplementation of BSA and FCS caused significant (P < 0.01) decreases in malondialdehyde (MDA) level at 48 h and 72 h, they significantly (P < 0.01) increased the glutathione peroxidase (GPx) antioxidant activity up to 72 h when compared to the control group. In conclusion, BSA and FCS supplementation to liquid stored rabbit semen provide a protection for spermatozoa against cool storage-induced DNA damage and plasma membrane integrity by their antioxidative properties.  相似文献   

15.
Temperature-controlled Atomic Force Microscopy (TC-AFM) in Contact Mode is used here to directly image the mechanisms by which melting and crystallization of supported, hydrated DPPC bilayers proceed in the presence and absence of the model peptide WALP23. Melting from the gel Lβ′ to the liquid-crystalline Lα phase starts at pre-existing line-type packing defects (grain boundaries) in absence of the peptide. The exact transition temperature is shown to be influenced by the magnitude of the force exerted by the AFM probe on the bilayer, but is higher than the main transition temperature of non-supported DPPC vesicles in all cases due to bilayer–substrate interactions. Cooling of the fluid Lα bilayer shows the formation of the line-type defects at the borders between different gel-phase regions that originate from different nuclei. The number of these defects depends directly on the rate of cooling through the transition, as predicted by classical nucleation theory.The presence of the transmembrane, synthetic model peptide WALP23 is known to give rise to heterogeneity in the bilayer as microdomains with a striped appearance are formed in the DPPC bilayer. This striated phase consists of alternating lines of lipids and peptide. It is shown here that melting starts with the peptide-associated lipids in the domains, whose melting temperature is lowered by 0.8–2.0 °C compared to the remaining, peptide-free parts of the bilayer. The stabilization of the fluid phase is ascribed to adaptations of the lipids to the shorter peptide. The lipids not associated with the peptide melt at the same temperature as those in the pure DPPC supported bilayer.  相似文献   

16.
Farnesol interacts with membranes in a wide variety of biological contexts, yet our understanding of how it affects lipid bilayers is not yet complete. This study investigates how the 15-carbon isoprenoid, farnesol, influences the phase behaviour, lateral organization, and mechanical stability of dimyristol phosphatidylcholine (DMPC) model membranes. Differential scanning calorimetry (DSC) of multilamellar DMPC-farnesol mixtures (up to 26 mol% farnesol) demonstrates how this isoprenoid lowers and broadens the gel-fluid phase transition. A gel-fluid coexistence region becomes progressively more dominant with increasing farnesol concentration and at concentrations of and greater than 10.8 mol%, an upper transition emerges at about 35 °C. Atomic force microscopy images of supported farnesol-DMPC bilayers containing 10 and 20 mol% farnesol provide structural evidence of gel-fluid coexistence around the main transition. Above this coexistence region, membranes exhibit homogeneous lateral organization but at temperatures below the main gel-fluid coexistence region, another form of phase coexistence is observed. The solid nature of the gel phase is confirmed using micropipette aspiration. The combined thermodynamic, structural, and mechanical data allow us to construct a phase diagram. Our results show that farnesol preferentially partitions into the fluid phase and induces phase coexistence in membranes below the main transition of the pure lipid.  相似文献   

17.
Membrane curvature remodeling induced by amphipathic helices (AHs) is essential in many biological processes. Here we studied a model amphipathic peptide, M2AH, derived from influenza A M2. We are interested in how M2AH may promote membrane curvature by altering membrane physical properties. We used atomic force microscopy (AFM) to examine changes in membrane topographic and mechanical properties. We used electron paramagnetic resonance (EPR) spectroscopy to explore changes in lipid chain mobility and chain orientational order. We found that M2AH perturbed lipid bilayers by generating nanoscale pits. The structural data are consistent with lateral expansion of lipid chain packing, resulting in a mechanically weaker bilayer. Our EPR spectroscopy showed that M2AH reduced lipid chain mobility and had a minimal effect on lipid chain orientational order. The EPR data are consistent with the surface-bound state of M2AH that acts as a chain mobility inhibitor. By comparing results from different lipid bilayers, we found that cholesterol enhanced the activity of M2AH in inducing bilayer pits and altering lipid chain mobility. The results were explained by considering specific M2AH-cholesterol recognition and/or cholesterol-induced expansion of interlipid distance. Both AFM and EPR experiments revealed a modest effect of anionic lipids. This highlights that membrane interaction of M2AH is mainly driven by hydrophobic forces. Lastly, we found that phosphatidylethanolamine (PE) lipids inhibited the activity of M2AH. We explained our data by considering interlipid hydrogen-bonding that can stabilize bilayer organization. Our results of lipid-dependent membrane modulations are likely relevant to M2AH-induced membrane restructuring.  相似文献   

18.
This review describes the application of fluorescence correlation spectroscopy (FCS) for the study of biological membranes. Monitoring the fluorescence signal fluctuations, it is possible to obtain diffusion constants and concentrations for several membrane components. Focusing the attention on lipid bilayers, we explain the technical difficulties and the new FCS-based methodologies introduced to overcome them. Finally, we report several examples of studies which apply FCS on both model and biological membranes to obtain interesting insight in the topic of lateral membrane organization.  相似文献   

19.
Interaction of electric fields with biological systems has begun to receive considerable attention for applications that include field-assisted drug delivery, medical interventions, and genetic engineering. External fields induce the strongest effects at membranes with electroporation being a common feature. Membrane transport in this context of poration is often based on continuum approaches utilizing macroscopic parameters such as the permittivity, diffusion coefficients, and mobilities. In such modeling, field dependences, local inhomogeneities, and microscopic details are usually ignored. Here, a molecular dynamics (MD) scheme is used for a more rigorous and physically realistic evaluation of such parameters for potential application to electroporative transport model development. A suitable membrane structure containing a nanopore derived from MD analysis is used as the initial geometric configuration. Both static and frequency dependent diffusion coefficients have been evaluated. Permittivities are also calculated and shown to be dramatically non-uniform in the vicinity of membranes under high external fields. A positive feedback mechanism leading to enhanced membrane fields is discussed.  相似文献   

20.
Representing a physiological “Achilles' heel”, the cell wall precursor lipid II (LII) is a prime target for various classes of antibiotics. Over the years LII-binding agents have been recognized as promising candidates and templates in the search for new antibacterial compounds to complement or replace existing drugs. To elucidate the molecular structural basis underlying LII functional mechanism and to better understand if and how lantibiotic binding alters the molecular behavior of LII, we performed molecular dynamics (MD) simulations of phospholipid membrane-embedded LII in the absence and presence of the LII-binding lantibiotic nisin. In a series of 2 × 4 independent, unbiased 100 ns MD simulations we sampled the conformational dynamics of nine LII as well as nine LII–nisin complexes embedded in an aqueous 150 mM NaCl/POPC phospholipid membrane environment. We found that nisin binding to LII induces a reduction of LII mobility and flexibility, an outward shift of the LII pentapeptide, an inward movement of the LII disaccharide section, and an overall deeper insertion of the LII tail group into the membrane. The latter effect might indicate an initial step in adopting a stabilizing, scaffold-like structure in the process of nisin-induced membrane leakage. At the same time nisin conformation and LII interaction remain similar to the 1WCO LII–nisin NMR solution structure.  相似文献   

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