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1.
Urška Batista Maja Garvas Marjana Nemec Milan Schara Peter Veranič Tilen Koklic 《Cell biology international》2010,34(6):663-668
Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V‐79 cells in the plateau phase of growth (arrested in G1). We have measured cell viability by a dye‐exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin‐probe MeFASL(10,3) (5‐doxylpalmitoyl‐methylester), which partitions mainly in cell membranes and the hydrophilic spin‐probe TEMPONE (4‐oxo‐2,2,6,6‐tetramethylpiperidine‐1‐oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin‐probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin‐treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V‐79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged. 相似文献
2.
Praper T Sonnen A Viero G Kladnik A Froelich CJ Anderluh G Dalla Serra M Gilbert RJ 《The Journal of biological chemistry》2011,286(4):2946-2955
Perforin (PFN) is a pore-forming protein produced by cytotoxic lymphocytes that aids in the clearance of tumor or virus-infected cells by a mechanism that involves the formation of transmembrane pores. The properties of PFN pores and the mechanism of their assembly remain unclear. Here, we studied pore characteristics by functional and structural methods to show that perforin forms pores more heterogeneous than anticipated. Planar lipid bilayer experiments indicate that perforin pores exhibit a broad range of conductances, from 0.15 to 21 nanosiemens. In comparison with large pores that possessed low noise and remained stably open, small pores exhibited high noise and were very unstable. Furthermore, the opening step and the pore size were dependent on the lipid composition of the membrane. The heterogeneity in pore sizes was confirmed with cryo-electron microscopy and showed a range of sizes matching that observed in the conductance measurements. Furthermore, two different membrane-bound PFN conformations were observed, interpreted as pre-pore and pore states of the protein. The results collectively indicate that PFN forms heterogeneous pores through a multistep mechanism and provide a new paradigm for understanding the range of different effects of PFN and related membrane attack complex/perforin domain proteins observed in vivo and in vitro. 相似文献
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Factor Xa (FXa) has a prominent role in amplifying both inflammation and the coagulation cascade. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Efficient proteolysis is well known to require phosphatidylserine (PS)-containing membranes that are provided by platelets in vivo. However, soluble, short-chain PS also triggers efficient proteolytic activity and formation of an inactive FXa dimer in solution. In this work, we ask whether PS-containing membranes also trigger formation of an inactive FXa dimer. We determined the proteolytic activity of human FXa toward human Pre2 as a substrate both at fixed membrane concentration (increasing FXa concentration) and at fixed FXa concentration (increasing membrane concentration). Neither of these experiments showed the expected behavior of an increase in activity as FXa bound to membranes, but instead suggested the existence of a membrane-bound inactive form of FXa. We found also that the fluorescence of fluorescein attached to FXa's active site serine was depolarized in a FXa concentration-dependent fashion in the presence of membranes. The fluorescence lifetime of FXa labeled in its active sites with a dansyl fluorophore showed a similar concentration dependence. We explained all these observations in terms of a quantitative model that takes into account dimerization of FXa after binding to a membrane, which yielded estimates of the FXa dimerization constant on a membrane as well as the kinetic constants of the dimer, showing that the dimer is effectively inactive. 相似文献
5.
We have investigated interaction of alkyphospholipid (APL) liposomes consisting of 1,1-dimethylpiperidin-1-ium-4-yl) octadecyl phosphate (OPP) and different concentrations of cholesterol (CH) with human MT-3 breast-cancer cells using electron paramagnetic resonance method (EPR) with advanced characterization of EPR spectra of spin labeled liposome membranes. After incubation of OPP liposomes with MT-3 cells, a reduction of liposome entrapped, water soluble spin-probe tempocholine (ASL) was observed, indicating that ASL is released from liposomes and is reduced by oxy-redoxy systems inside the cells. This process is fast if cholesterol content in the bilayer was 29 or 45 mol%, whereas at 56 mol% cholesterol the process is almost stopped. The rate of spin-probe reduction in first 10 min after incubation with cells is even faster as for the free ASL, indicating that liposomes with low amount of cholesterol accelerate penetration of ASL into the cells. A faster release of hydrophilic material from liposomes with low cholesterol content coincides with the presence of domains with highly disordered alkyl chain motion that disappears at 50 mol% of cholesterol. We propose that these highly fluid domains are responsible for interaction of OPP liposomes with cells and fast release of the entrapped material into the cells. These results suggest that micelles are not the only reason for cytotoxic effect of OPP liposome formulations, as it was suggested before. OPP in liposomes, containing 45 mol% cholesterol or less, also contributes to the cytotoxic effect, due to their fast interaction with breast-cancer cells. 相似文献
6.
Life-cycle assessment of a hydrogen-based uninterruptible power supply system using renewable energy
Mitja Mori Miha Jensterle Tilen Mržljak Boštjan Drobnič 《The International Journal of Life Cycle Assessment》2014,19(11):1810-1822
Purpose
The paper provides an empirical assessment of an uninterruptible power supply (UPS) system based on hydrogen technologies (HT-UPS) using renewable energy sources (RES) with regard to its environmental impacts and a comparison to a UPS system based on the internal combustion engine (ICE-UPS).Methods
For the assessment and comparison of the environmental impacts, the life-cycle assessment (LCA) method was applied, while numerical models for individual components of the UPS systems (electrolyser, storage tank, fuel cell and ICE) were developed using GaBi software. The scope of analysis was cradle-to-end of utilisation with functional unit 1 kWh of uninterrupted electricity produced. For the life-cycle inventory analysis, quantitative data was collected with on-site measurements on an experimental system, project documentation, GaBi software generic databases and literature data. The CML 2001 method was applied to evaluate the system’s environmental impacts. Energy consumption of the manufacturing phase was estimated from gross value added (GVA) and the energy intensity of the industry sector in the manufacturer’s country.Results and discussion
In terms of global warming (GW), acidification (A), abiotic depletion (AD) and eutrophication (E), manufacturing phase of HT-UPS accounts for more than 97 % of environmental impacts. Electrolyser in all its life-cycle phases contributes above 50 % of environmental impacts to the system’s GW, A and AD. Energy return on investment (EROI) for the HT-UPS has been calculated to be 0.143 with distinction between renewable (roughly 60 %) and non-renewable energy resources inputs. HT-UPS’s life-cycle GW emissions have been calculated to be 375 g of CO2 eq per 1 kWh of uninterruptible electric energy supplied. All these values have also been calculated for the ICE-UPS and show that in terms of GW, A and AD, the ICE-UPS has bigger environmental impacts and emits 1,190 g of CO2 eq per 1 kWh of uninterruptible electric energy supplied. Both systems have similar operation phase energy efficiency. The ICE-UPS has a higher EROI but uses almost none RES inputs.Conclusions
The comparison of two different technologies for providing UPS has shown that in all environmental impact categories, except eutrophication, the HT-UPS is the sounder system. Most of HT-UPS’s environmental impacts result from the manufacturing phase. On the contrary, ICE-UPS system’s environmental impacts mainly result from operational phase. Efficiency of energy conversion from electricity to hydrogen to electricity again is rather low, as is EROI, but these will likely improve as the technology matures. 相似文献7.
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Zeisig R Koklic T Wiesner B Fichtner I Sentjurc M 《Archives of biochemistry and biophysics》2007,459(1):98-106
To study whether membrane fluidity of tumor cells have an influence on metastasis, MT3 breast cancer cells harvested during exponential growth and under confluent conditions were compared. Electron paramagnetic resonance (EPR) data revealed that, in comparison to growing cells, confluent cells have a significant higher fluidity in their membrane related to a higher relative portion of disordered domains and a reduced portion of the most ordered domains. Further, sialyl Lewis X and/or A ligand-mediated adhesion of these cells was 2-fold enhanced. Confocal laser scanning microscopy further demonstrated a higher motility of ligands in the membrane of confluent cells, together with an accumulation of these ligands in distinct areas. Both facts are suggested to be responsible for an enhanced cell adhesion observed. Finally, an increased number of large distinct metastatic foci was registered in lungs of mice after i.v. inoculation of confluent cells. The results indicate that domain organization and fluidity of the cell membrane affect tumor cell adhesion and can have in this way also an impact on the malignancy of breast cancer cells. 相似文献
9.
Haque ME Chakraborty H Koklic T Komatsu H Axelsen PH Lentz BR 《Biophysical journal》2011,(5):1095-1104
While the importance of viral fusion peptides (e.g., hemagglutinin (HA) and gp41) in virus-cell membrane fusion is established, it is unclear how these peptides enhance membrane fusion, especially at low peptide/lipid ratios for which the peptides are not lytic. We assayed wild-type HA fusion peptide and two mutants, G1E and G13L, for their effects on the bilayer structure of 1,2-dioleoyl-3-sn-phosphatidylcholine/1,2-dioleoyl-3-sn-phosphatidylethanolamine/Sphingomyelin/Cholesterol (35:30:15:20) membranes, their structures in the lipid bilayer, and their effects on membrane fusion. All peptides bound to highly curved vesicles, but fusion was triggered only in the presence of poly(ethylene glycol). At low (1:200) peptide/lipid ratios, wild-type peptide enhanced remarkably the extent of content mixing and leakage along with the rate constants for these processes, and significantly enhanced the bilayer interior packing and filled the membrane free volume. The mutants caused no change in contents mixing or interior packing. Circular dichroism, polarized-attenuated total-internal-reflection Fourier-transform infrared spectroscopy measurements, and membrane perturbation measurements all conform to the inverted-V model for the structure of wild-type HA peptide. Similar measurements suggest that the G13L mutant adopts a less helical conformation in which the N-terminus moves closer to the bilayer interface, thus disrupting the V-structure. The G1E peptide barely perturbs the bilayer and may locate slightly above the interface. Fusion measurements suggest that the wild-type peptide promotes conversion of the stalk to an expanded trans-membrane contact intermediate through its ability to occupy hydrophobic space in a trans-membrane contact structure. While wild-type peptide increases the rate of initial intermediate and final pore formation, our results do not speak to the mechanisms for these effects, but they do leave open the possibility that it stabilizes the transition states for these events. 相似文献
10.
Marlon Lawrence Anze Testen Tilen Koklic Oliver Smithies 《Journal of visualized experiments : JoVE》2016,(108)
Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated >300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy. 相似文献