Enhancement of lysosomal osmotic sensitivity induced by the photooxidation of membrane thiol groups

The osmotic lysis of photodamaged lysosomes is a critical event for killing tumor cells. How the photodamage increases lysosomal osmotic sensitivity is still unclear. In this work, the effect of the photooxidation of membrane thiol groups on the lysosomal osmotic sensitivity was studied by measuring the thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid) and examining the lysosomal beta-hexosaminidase latency loss in a hypotonic sucrose medium. The results show that methylene blue-mediated photooxidation of lysosomes decreased their membrane thiol groups and produced cross-linkage of membrane proteins (molecular weight ranging from 75000 to 125000), which was visualized by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Simultaneously, the lysosomal osmotic sensitivity increased. These photoinduced alterations of the lysosomes could be recovered by reducing the oxidized thiol groups with dithiothreitol. It indicates that the photooxidation of membrane thiol groups can increase the lysosomal osmotic sensitivity and therefore provides a new explanation for the photoinduced lysosomal lysis.     

The effects of polymer chain rigidification, zeolite pore size and pore blockage on polyethersulfone (PES)-zeolite A mixed matrix membranes

The polyethersulfone (PES)-zeolite 3A, 4A and 5A mixed matrix membranes (MMMs) were fabricated with a modified solution-casting procedure at high temperatures close to the glass transition temperatures (T_g) of polymer materials. The effects of membrane preparation methodology, zeolite loading and pore size of zeolite on the gas separation performance of these mixed matrix membranes were studied. SEM results show the interface between polymer and zeolite in MMMs experiencing natural cooling is better (i.e., less defective) than that in MMMs experiencing immediate quenching. The increment of glass transition temperature (T_g) of MMMs with zeolite loading confirms the polymer chain rigidification induced by zeolite. The experimental results indicate that a higher zeolite loading results in a decrease in gas permeability and an increase in gas pair selectivity. The unmodified Maxwell model fails to correctly predict the permeability decrease induced by polymer chain rigidification near the zeolite surface and the partial pore blockage of zeolites by the polymer chains. A new modified Maxwell model is therefore proposed. It takes the combined effects of chain rigidification and partial pore blockage of zeolites into calculation. The new model shows much consistent permeability and selectivity predication with experimental data. Surprisingly, an increase in zeolite pore size from 3 to 5 Å generally not only increase gas permeability, but also gas pair selectivity. The O₂/N₂ selectivity of PES-zeolite 3A and PES-zeolite 4A membranes is very similar, while the O₂/N₂ selectivity of PES-zeolite 5A membranes is much higher. This implies the blockage may narrow a part of zeolite 5A pores to approximately 4 Å, which can discriminate the gas pair of O₂ and N₂, and narrow a part of zeolites 3A and 4A pores to smaller sizes. It is concluded that the partial pore blockage of zeolites by the polymer chains has equivalent or more influence on the separation properties of mixed matrix membranes compared with that of the polymer chain rigidification.     

Spin label studies on rat liver and heart plasma membranes: effects of temperature, calcium, and lanthanum on membrane fluidity.

The structures of rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(12,3). The polarity-corrected order parameters (S) of liver and heart plasma membranes were independent of probe concentration only if experimentally determined low I(12,3)/lipid ratios were employed. At higher probe/lipid ratios, the order parameters of both membrane systems decreased with increasing probe concentration, and these effects were attributed to enhanced nitroxide radical interactions. Examination of the temperature dependence of approximate and polarity-corrected order parameters indicated that lipid phase separations occur in liver (between 19 degrees and 28 degrees C) and heart (between 21 degrees and 32 degrees C) plasma membranes. The possibility that a wide variety of membrane-associated functions may be influenced by these thermotropic phase separations is considered. Addition of 3.9 mM CaCl2 to I(12,3)-labeled liver plasma membrane decreased the fluidity as indicated by a 5% increase in S at 37 degrees C. Similarly, titrating I(12,3)-labeled heart plasma membranes with either CaCl2 or LaCl3 decreased the lipid fluidity at 37 degrees C, although the magnitude of the La3+ effect was larger and occurred at lower concentrations than that induced by Ca2+; addition of 0.2 mM La3+ or 3.2 mM Ca2+ increased S by approximately 7% and 5%, respectively. The above cation effects reflected only alterations in the membrane fluidity and were not due to changes in probe--probe interactions. Ca2+ and La3+ at these concentrations decrease the activities of such plasma membrane enzymes as Na+, K+-ATPase and adenylyl cyclase, and it is suggested that the inhibition of these enzymes may be due in part to cation-mediated decreases in the lipid fluidity.     

Promotion of Proapoptotic Signals by Lysosomal Photodamage

We previously reported that low‐level lysosomal photodamage enhanced the efficacy of subsequent mitochondrial photodamage, resulting in a substantial promotion of apoptotic cell death. We now extend our analysis of the sequential PDT protocol to include two additional lysosomal‐targeting photosensitizers. These agents, because of enhanced permeability, are more potent than the agent (N‐aspartyl chlorin E6, NPe6) used in the initial study. Addition of the cell‐permeable cysteine protease inhibitor E‐64d and calcium chelator BAPTA‐AM almost completely suppressed sequential PDT‐induced loss of mitochondrial membrane potential and activation of procaspases‐3 and ‐7. These inhibitors did not, however, suppress the proapoptotic effect of a BH3 mimetic or mitochondrial photodamage. Knockdowns of ATG7 or ATG5, proteins normally associated with autophagy, suppressed photodamage induced by the sequential PDT protocol. These effects appear to be independent of the autophagic process as pharmacological inhibition of autophagy offered no such protection. Effects of ATG7 and ATG5 knockdowns may reflect the role that ATG7 plays in regulating lysosome permeability, and the likelihood that a proteolytic fragment of ATG5 amplifies mitochondrial proapoptotic processes. Our results suggest that low‐dose photodamage that sequentially targets lysosomes and mitochondria may offer significant advantages over the use of single photosensitizers.     

Ultrastructural damage in photosensitized endothelial cells: dependence on hematoporphyrin delivery pathways.

The subcellular photodamage to endothelial cells in culture, revealed by transmission electron microscopy, was correlated with discrete delivery pathways of hematoporphyrin (HP). Cell detachment from the extracellular matrix, prominent water influx starting at the outer membrane and formation of blebs followed by cell death were the result of photodynamic damage induced by aqueous HP. Serum-bound HP was internalized by endocytosis and accumulated in lysosomal compartments as located after photosensitization. Obstructed lysosomal membranes, degradation of chromatin and swelling of endoplasmic reticulum were revealed in these cells. Red blood cells (RBCs), preincubated with HP, delivered low amounts of the drug to endothelial cells. The photodamage was limited to the nucleus and nucleolus. The role of photosensitizer delivery pathways in cancer cell damage is discussed.     

Determinants of the apoptotic response to lysosomal photodamage

Studies with mouse leukemia L1210 cells revealed that selective lysosomal photodamage caused by any of three photosensitizing agents was followed by a gradual loss of the mitochondrial membrane potential (delta psi m), release of cytochrome c into the cytosol, increased DEVDase activity (a measure of levels of caspase-3) and a limited apoptotic response. Similar effects were observed in the murine hepatoma 1c1c7 cell line. Immunofluorescence techniques employing 1c1c7 cells demonstrated the immediate release of the lysosomal enzyme cathepsin B following lysosomal photodamage. These studies suggest that the cytotoxic effects of lysosomal photodamage are initiated by released lysosomal proteases that either directly and/or indirectly activate caspases as a consequence of the induction of mitochondrial damage.     

Use of ionic membranes in the fractionation of water-alcohol mixtures by the pervaporation process

Five membranes, constituted of ionic chains grafted onto inert matrix, were studied in pervaporation of water - ethanol mixtures of different compositions. The permeability and selectivity of all five membranes showed a marked dependence on the nature of the counter-ions. Water permeates preferentially through all the types of membrane and counter-ions, except the protonated carboxylate membrane. For cation exchange membranes with alcaline counter-ions, the selectivity decreases in the following sequence: K+<Na+<Li+<H+. The permeability of the sulfonate membrane follows the reversed sequence, while that of the carboxylate membrane follows the same sequence, i.e. there is no trade-off between permeability and selectivity for the latter membrane. The anion-exchange (quaternized amine) membrane behaved similarly to the sulfonate membrane, i.e. the permeability and the selectivity vary in opposite directions. An attempt to interpret the influence of the nature of counter-ions on the basis of physico-chemical properties of the ion pairs was made.     

PHOTOSENSITIZATION WITH ETIOBENZOCHLORINS AND OCTAETHYLBENZOCHLORINS

The photophysical and photobiological properties of a series of etiobenzochlorins were evaluated in cell culture using murine leukemia L1210 cells. In the series of agents tested, the chlorin-(mono)sulfonate was the most efficacious, the tin chlorin somewhat less so and the tin chlorin-sulfonate much less active. The parent chlorin was essentially inactive at the limit of solubility. Photodamage was assessed by measuring alterations in surface hydro-phobicity ( via a two-phase partitioning procedure), amino acid transport and membrane potential. Additional information was provided from fluorescence microscopy, which was used to identify sites of sensitizer binding and effects of photodamage on the binding patterns of fluorescent probes specific for mitochondria, lysosomes and plasma membranes. Effects of photodamage on fluorescence lifetime distribution of the membrane probe trimethylamino-diphenyl hexatriene were examined. The data obtained were consistent with localization of the parent etiobenzochlorin and tin derivative at lysosomal loci. the chlorin-sulfonate at plasma and mitochondrial membranes and tin-sulfonate at the cell surface.     

EPR study of the effect, induced by zidovudine (AZT), on the membrane lipid dynamics in leukemic cell

Zidovudine (AZT) was the first drug approved by the FDA for treatment of HIV infections. To investigate the AZT effects on the physical properties of K562 cell membranes, experiments were performed by measuring an order parameter value in these cells, previously labelled with 16-DSA (16-doxyl-stearic acid) or 5-DSA (5-doxyl-stearic acid). The EPR spectra of the labelled fatty acid were used to detect the alteration in their freedom of motion and to provide indications of membrane fluidity. Two different parameters were calculated from EPR spectra for the 16-DSA and 5-DSA. Preliminary data indicate that, for both the probes used, these parameters were not significantly different in the control cells with respect to the AZT-treated ones. Control measurements, performed to test the sensitivity of the technique using the DMSO agent, showed a significant increase in the K562 membrane fluidity.     

聚乙烯醇缩醛膜的加湿透气性

前曾报道聚乙烯醇(PVA)与脂肪醛缩合后亲水性的羟基减少,影响到其薄膜的透湿性和透气性,本文考察PVA及其三种脂肪族缩醛物膜在加湿条件下的透气性。 PVA膜为10％PVA水溶液用流涎法制成。缩醛膜为PVA缩醛衍生     

Fluorescence studies on radiation oxidative damage to membranes with implications to cellular radiosensitivity

Radiation oxidative damage to plasma membrane and its consequences to cellular radiosensitivity have received increasing attention in the past few years. This review gives a brief account of radiation oxidative damage in model and cellular membranes with particular emphasis on results from our laboratory. Fluorescence and ESR spin probes have been employed to investigate the structural and functional alterations in membranes after y-irradiation. Changes in the lipid bilayer in irradiated unilamellar liposomes prepared from egg yolk lecithin (EYL) were measured by using diphenylhexatriene (DPH) as a probe. The observed increase in DPH polarization and decrease in fluorescence intensity after γ-irradiation of liposomes imply radiation-induced decrease in bilayer fluidity. Inclusion of cholesterol in liposome was found to protect lipids against radiation damage, possibly by modulation of bilayer organization e.g. lipid packing. Measurements on dipalmitoyl phosphatidylcholine (DPPC) liposomes loaded with 6-carboxyfluorescein (CF) showed radiation dose-dependent release of the probe indicating radiation-induced increased permeability. Changes in plasma membrane permeability of thymocytes were monitored by fluorescein diacetate (FDA) and induced intracellular reactive oxygen species (ROS) were determined by 2,7-dichlorodihydro fluorescein diacetate (DCH-FDA). Results suggest a correlation between ROS generation and membrane permeability changes induced by radiation within therapeutic doses (0-10 Gy). It is concluded that increase in membrane permeability was the result of ROS-mediated oxidative reactions, which might trigger processes leading to apoptotic cell death after radiation exposure.     

Light-regulated permeability of rhodopsin-phospholipid membrane vesicles.

Abstract—Light absorption by rhodopsin in receptor cell membranes initiates the excitation of the receptor cell. Rhodopsin-phospholipid membrane vesicles were studied to localize initial transduction events. Rhodopsin-phospholipid recombinant membranes are thermally stable and light sensitive and may be chemically regenerated after bleaching in the same manner as receptor cell membranes. Rhodopsin-containing vesicles prepared from unsaturated phosphatidylcholine (PCho) or PCho and phosphatidylethanolaminc display kinetics for the metarhodopsin I to II transition which are comparable to those of receptor cell membranes. NMR spectroscopy was used to examine the permeability of the membrane vesicles to added shift (Eu³⁺) or relaxation reagents (Mn²⁺, Co²⁺). Unexposed rhodopsin-phospholipid vesicles are sealed to ion movement and become permeable after light exposure. Selected ions (Ca²⁺, Mn²⁺, Co²⁺) may be photoreleased from the interior of loaded membrane vesicles. The quantity released is proportional to the initial ionic concentration. The number of ions released/rhodopsin bleached is dependent on the light intensity, and high yields (40–160) of Ca²⁺/rhodopsin bleached are observed at low levels of light bleaching. The present results indicate that rhodopsin spans the phospholipid bilayer membrane, and are consistent with an increase in the permeability of the membrane initiated by light excitation of rhodopsin.     

Effects of light and cGMP on membrane fluidity of frog rod outer segments.

Abstract—We have used a spin-labeled fatty acid to detect changes induced by light and by cGMP in isolated rod outer segment membranes. We chose a spin probe (5-doxylstearic acid) which has the nitroxide group placed on the hydrocarbon chain, so the probe should reside somewhat inside the hydrophobic region of the membrane. We found that light exposures which bleached the rhodopsin also produced a small change in the EPR spectra. The spectral changes are consistent with a small increase in membrane fluidity. Light exposures which bleach rhodopsin are known to activate a phosphodiesterase that markedly decreases the cGMP level in rod outer segment. Therefore, we attempted to vary cGMP levels directly by adding Bu₂-cGMP, or indirectly, by adding IBMX, CDTA or ATP to try to inhibit the phosphodiesterase. In each case where the cGMP level is expected to increase, we observed spectral changes in the dark which suggested a small decrease in membrane fluidity. Thus, all of our results with this probe are consistent with the idea that changing the level of cGMP produces changes in membrane fluidity. The light-induced spectral changes we observed required the presence of ATP, and were inhibited by 2mM Ca²⁺, or by the chelator of divalent cations, CDTA.     

Transport through zeolite filled polymeric membranes

In this paper the effect of zeolite particles incorporated in rubbery polymers on the pervaporation properties of membranes made from these polymers is discussed. Pervaporation of methanol/toluene mixtures was carried out with membranes prepared from the toluene selective polymer EPDM and the methanol selective polymers Viton and Estane 5707. From the results of the pervaporation experiments it could be concluded that the addition of the hydrophilic zeolite NaX as well as the hydrophobic zeolite silicalite-1 leads to an increase in methanol flux and a decrease in toluene flux through the membranes. Pervaporation experiments with bi-layer membranes consisting of an unfilled polymer layer filled with zeolite particles demonstrated that the effect of addition of particles depends on their position in the membrane. Furthermore, the component flux through the membranes as a function of the volume fraction of zeolite is modelled with existing theories describing the permeability of heterogeneous materials. The results show that the apparent permeability of the dispersed phase is lower than the intrinsic permeability of the dispersed phase when the flux through the particle is restricted by the polymer phase. This phenomenon was confirmed by numerical simulation of the transport in the membrane through a plane parallel to the transport direction. The simulations are carried out for an unfilled membrane, a membrane filled with an impermeable particle, a rubber particle and with a particle which shows Langmuir sorption behaviour. The reason for the discrepancy between the apparent permeability and the intrinsic permeability is that the apparent permeability of the zeolite phase is calculated by dividing the flux with the driving force over the entire membrane which is larger than that over the particle. In case of numerical simulation the concentration in every position in the plane is known and therefore the intrinsic permeability of the filler can be calculated on basis of the actual driving force. This treatment results in a permeability which is correct over several orders of magnitude.     

Lysosomes: a possible target for psoralen photodamage

Treatment in vitro of Ehrlich ascites tumor cells or human fibroblasts with 8-methoxypsoralen (8-MOP, 2.4 microM) and UVA irradiation results in a 30% and 60% respectively reduction in lysosomal beta-galactosidase activity in situ. Under identical conditions one 8-MOP adduct was formed per 2 X 10(4) bases of DNA, one 8-MOP adduct was formed per approximately 10(4) tRNA molecules and one per approximately 100 ribosomes. It is suggested that the decrease in lysosomal beta-galactosidase activity is a result of leakage through the lysosomal membrane caused by psoralen-UVA damage of the lipids in the membrane, since no effect was found on beta-galactosidase in vitro. These results indicate that the lysosomes may also be a target for cellular photodamage by 8-methoxy-psoralen.     

Gas permeation through water-swollen hydrogel membranes

This work deals with water-swollen hydrogel membranes for potential CO₂ separation applications, with an emphasis on elucidating the role of water in the membrane for gas permeation. A series of hydrogel membranes with a wide range of water contents (0.9–10 g water/g polymer) were prepared from poly(vinyl alcohol), chitosan, carboxyl methyl cellulose, alginic acid and poly(vinylamine), and the permeation of CO₂, H₂, He and N₂ through the membranes at different pressures (200–800 kPa) was studied. The gas permeabilities through the dry dense membranes were measured as well to evaluate the resistance of the polymer matrix in the hydrogel membranes. It was shown that the gas permeability in water-swollen membrane is lower than the gas permeability in water, and the selectivity of the water-swollen membranes to a pair of gases is close to the ratios of their permeabilities in water. The permeability of the water-swollen membranes increases with an increase in the swelling degree of the membrane, and the membrane permeability tends to level off when the water content is sufficiently high. A resistance model was proposed to describe gas permeation through the hydrogel membranes, where the immobilized water retained in the polymer matrix was considered to form transport passageways for gas permeation through the membrane. It was shown that the permeability of hydrogel membranes was primarily determined by the water content in the membrane. The model predictions were consistent with the experimental data for various hydrogel membranes with a wide range of water contents (0.4–10 g water/g polymer).     

The effect of tourmaline on cell membrane of nitrosomonas europaea and biodegradation of micropollutant

As a kind of ammonia‐oxidizing bacteria, Nitrosomonas europaea (N. europaea) was chosen as a research model to study the alteration of cell membrane in the presence of tourmaline and biodegradation of acetochlor. atomic force microscopy images reveal that the presence of tourmaline substantially changes the structure of the outer membrane of the cell responsible for the cell permeability. SEM images show that the introduction of tourmaline makes the cell lose its ability to resist lysozyme owing to the damages. The fluorescence polarization has shown a significant decrease in membrane fluidity and the increase of permeability of cell membrane. Ca²⁺ and Mg²⁺ was measured using inductively coupled plasma mass spectrometry and was found in the supernatant from the cells treated by tourmaline. Tourmaline can improve the efficiency of biodegradation of acetochlor for N. europaea. It is proposed that the cell permeability is slightly increased, and the absorbability of nutrition from the medium becomes easier. As a result, N. europaea grows faster in the presence of tourmaline than the native cells. Copyright © 2014 John Wiley & Sons, Ltd.     

Optimization study of polyethylene glycol and solvent system for gas permeation membranes

Cellulose acetate (CA) membranes blended with Polyethylene glycol (PEG) in acetone–water solvent system were synthesized by using solution-casting method that resulted in the formation of flexible, white membranes. Different molecular weight (MW) grades of PEG (including MW 1000, 10,000 and 20,000?g/mol) were used. Cast membranes were tested for tensile strength and permeability at different loading of PEG MW 10,000 and 20,000?g/mol. Excellent flexible membranes were produced in acetone–water solvent system in the presence of PEG, which were otherwise brittle. Surface structure and morphology were analysed using scanning electron microscopy. The presence of different functional groups was confirmed using Fourier transform infra-red spectroscopy and the mechanical characteristics were studied by tensile testing. The introduction of PEG caused an increase in permeability of the membranes. The increase in permeability is due to the opening up of pores as the membrane becomes more flexible, when the plasticizer is added. The permeability continues to increase with the addition of PEG. Moreover, the resulting membranes are not only more flexible, but also have largely improved tensile strength as compared to the CA membranes without PEG. This improved tensile strength can also be attributed to the improved flexibility of the membrane. A trade-off is reached between tensile strength and permeability as increasing amount of PEG improves tensile strength but the resulting membrane becomes too permeable to be used for gas separation. Moreover, using PEG of higher MW resulted in porous membranes, even at low amounts of PEG. Therefore, we concluded that CA membrane with less amount of low-MW PEG (i.e. 5% PEG of MW 1000?g/mol) must be used to optimize both permeability and tensile strength of the membrane.     

Thermo- and pH-responsive polypropylene microporous membrane prepared by the photoinduced RAFT-mediated graft copolymerization

Thermo- and pH-responsive polypropylene microporous membrane prepared by photoinduced reversible addition–fragmentation chain transfer (RAFT) graft copolymerization of acrylic acid and N-isopropyl acrylamide by using dibenzyltrithiocarbonate as a RAFT agent. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR/FT-IR), X-ray photoelectron spectroscopy (XPS) and field emission scanning electron microscopy (FE-SEM) were used to characterize the structural and morphological changes on the membrane surface. Results of ATR/FT-IR and XPS clearly indicated that poly(acrylic acid) (PAAc) and poly(N-isopropyl acrylamide) (PNIPAAm) were successfully grafted onto the membrane surface. The grafting chain length of PAAc on the membrane surface increased with the increase of UV irradiation time, and decreased with the increase of the concentration of chain transfer agent. The PAAc grafted membranes containing macro-chain transfer agents, or the living membrane surfaces were further functionalized via surface-initiated block copolymerization with N-isopropyl acrylamide in the presence of free radical initiator, 2,2′-azobisisobutyronitrile. It was found that PNIPAAm can be grafted onto the PAAc grafted membrane surface. The results demonstrated that polymerization of AAc and NIPAAm by the RAFT method could be accomplished under UV irradiation and the process possessing the living character. The PPMMs with PAAc and PNIPAAm grafting chains exhibited both pH- and temperature-dependent permeability to aqueous media.