Liposomal membrane fusion induced by extrusion method / 国际生物医学工程杂志

Objective To study the application of extrusion method in inducing liposome membrane fusion or membrane component mixing,and to investigate the effect of different extrusion conditions on liposome membrane fusion rate.Methods N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl) phosphatidylethanolamine (N-Rh-PE) labeled 1,2-dioleoyl lecithin (DOPC) liposomes and non-fluorescently labeled DOPC monolayer liposomes were mixed and extruded.The fluorescence changes before and after the extrusion of the mixed liposomes were observed using laser scanning confocal microscope,and the membrane fusion rate of the mixed liposomes was calculated by fluorescence resonance energy transfer method.Besides,the effects of extrusion times,extrusion pressure and temperature on the fusion rate of liposome membrane were studied.Results The results of laser scanning confocal microscopy showed that the distribution density and intensity of the green fluorescence of N-NBD-PE increased significantly after the extrusion of fluorescently labeled and non-fluorescent labeled DOPC liposomes,which confirmed membrane fusion.After 75 times of extrusion treatments,the liposome membrane fusion rate can reach 26％.The number of extrusions,extrusion pressure and temperature had a significant effect on the fusion rate of the liposome membrane.The higher the number of the extrusions,the smaller the extrusion pressure and the higher the efficiency of the liposome membrane fusion were at physiological temperature.Conclusions Extrusion method can induce liposome membrane fusion and membrane component mixing,and the prepared liposome has a narrower particle size distribution,which is expected to be a new method to induce the bilayer membrane fusion of liposome or lipid vesicle.

The role of C-terminus carbohydrate-binding domain of Vibrio cholerae haemolysin/cytolysin in the conversion of the pre-pore β-barrel oligomer to a functional diffusion channel.

Background & objectives: Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa pore-forming toxin (PFT) secreted by O1 El Tor and non-O1 strains. The purified toxin, which contains two C-terminus carbohydrate-binding domains in addition to the cytolytic domain at the core, causes lysis of a wide spectrum of eukaryotic cells at picomolar concentrations, apoptogenesis of intestinal and immune cells and accumulation of fluid in rabbit ligated ileal loop. Therefore, it may potentially complement the action of cholera toxin (CT) in diarrheagenic strains that do not produce CT. We showed earlier that β1-galactosyl-terminated glycoconjugates are strong inhibitors of its pore-forming activity, though carbohydrates are not functional receptors of VCC. Here, we investigate how the 15 kDa C-terminus β-prism lectin domain contributed to pore formation in erthrocytes. Methods: VCC was isolated from the culture supernatant of late log phase grown bacteria and purified to homogeneity by chromatography. The 50 kDa truncated variant was generated by restricted proteolysis. Liposome was prepared by sonication of a suspension of phospholipids and calceine release assay was done by spectrofluorometric monitoring of the released dye trapped in liposome. Formation of β-barrel oligomers in erythrocyte stroma was monitored by scanning electron microscopy. Results: Proteolytic truncation of the C-terminus β-prism lectin domain decreased hemolytic activity of the toxin by ~800-fold without causing a significant change in pore-forming activity toward synthetic lipid vesicles devoid of incorporated glycoproteins/glycolipids. Truncation at the C-terminus did not impair membrane-binding or assembly to the oligomeric pore. Interpretation & conclusions: Our data indicated that the C-terminus domain played a critical role in translocation of the pre-pore oligomeric assembly from the cell surface or lipid-water interface to the hydrocarbon core of the membrane bilayer, signaling the formation of functional diffusion channels.

Mechanisms of Selective Antimicrobial Activity of Gaegurin 4

Gaegurin 4 (GGN4), an antimicrobial peptide isolated from a Korean frog, is five times more potent against Gram-positive than Gram-negative bacteria, but has little hemolytic activity. To understand the mechanism of such cell selectivity, we examined GGN4-induced K+ efflux from target cells, and membrane conductances in planar lipid bilayers. The K+ efflux from Gram-positive M. luteus (2.5microgram/ml) was faster and larger than that from Gram-negative E. coli (75microgram/ml), while that from RBC was negligible even at higher concentration (100microgram/ml). GGN4 induced larger conductances in the planar bilayers which were formed with lipids extracted from Gram-positive B. subtilis than in those from E. coli (p<0.01), however, the effects of GGN4 were not selective in the bilayers formed with lipids from E. coli and red blood cells. Addition of an acidic phospholipid, phosphatidylserine to planar bilayers increased the GGN4-induced membrane conductance (p<0.05), but addition of phosphatidylcholine or cholesterol reduced it (p<0.05). Transmission electron microscopy revealed that GGN4 induced pore-like damages in M. luteus and dis-layering damages on the outer wall of E. coli. Taken together, the present results indicate that the selectivity of GGN4 toward Gram-positive over Gram-negative bacteria is due to negative surface charges, and interaction of GGN4 with outer walls. The selectivity toward bacteria over RBC is due to the presence of phosphatidylcholine and cholesterol, and the trans-bilayer lipid asymmetry in RBC. The results suggest that design of selective antimicrobial peptides should be based on the composition and topology of membrane lipids in the target cells.

Epidermal Barrier in Human Fetus and Newborn / 대한피부과학회지

BACKGROUND: The lipids of the stratum corneum, which originate from polar lipid precursors provided by the cells of the stratum granulosum via the exocytosis of lamellar bodies, with cornified cell envelope form competent epidermal barrier structurally and functionally. The ontogeny of the epidermal barrier is not clearly defined because of difficulty of sampling and methodology which defines epidermal lipids. OBJECT: From ultrastructural observation of skin samples obtained from human fetuses and newborn on serial developmental timings, we tried to clarify the sequential development of epidermal barrier. METHODS: Skin samples were obtained from 13 human fetuses from EGA(estimated gestational age) 10 to 23wks and 2 newborns. Specimens were observed by fluorescent confocal microscopy with nile red to identify the distribution of epidermal lipids, by transmission electron microscope with lanthanum to investigate the functional permeability barrier, with RuO4 to observe the intercellular lipid bilayer and morphology of lamellar bodies, with ion capture cytochemistry to investigate the formation of epidermal calcium gradient. RESULT: In nile red stain, the amount of epidermal lipid increased during fetal period. At EGA 23wks, the lipid distribution revealed linear and continuous pattern. In lanthanum tracer study, the electron dense tracer permeated all the intercellular space of the epidermis up to periderm and subepidermal space until EGA 21wks. At EGA 23wks, the tracer permeated intercellular space of epidermis weakly. It might be predicted that incomplete epidermal barrier is present at this time. In RuO4 stain, precursor of lamellar body was observed at EGA 15wks, and intercellular lipid bilayer was observed at EGA 16wks. As gestation increases, there was a steady increase in epidermal lipid bilayers. In ion capture cytochemistry, epidermal calcium gradient was first observed in follicular epidermis at EGA 20wks, and in interfollicular epidermis at EGA 23wks. From these results, it is concluded that the basic structures of epidermal barrier are formed at EGA 23wks, but it is not complete, and epidermal barrier arises first from follicular epidermis.

Effects of t-butyl hydrogen peroxide on single SR calcium release channels

Using lipid bilayer reconstitution technique, we investigated the oxidation effect of t-butyl hydrogen peroxide (tBHP) on the single channel activity of the sarcoplasmic reticulum (SR) calcium release channels isolated from canine latissimus dorsi muscles. When 0.7% tBHP was added in the cytosolic side, the channel activity became suppressed (n = 7), and it was recovered by changing the solution to the control solution. The suppression was due to the change in the gating mode of the channel: before tBHP the channel opened to four sub-conductance levels, but it opened to only one level after tBHP. These effects by tBHP were different from the previous finding using hydrogen peroxide (H2O2), which may be explained by different oxidation patterns between the two oxidants.

Increased activity of large conductance Ca2+-activated K+ channels in negatively-charged lipid membranes

The effects of membrane surface charge originated from lipid head groups on ion channels were tested by analyzing the activity of single large conductance Ca2+-activated K+ (maxi K) channel from rat skeletal muscle. The conductances and open-state probability (Po) of single maxi K channels were compared in three types of planar lipid bilayers formed from a neutral phosphatidyledianolamine (PE) or two negatively-charged phospholipids, phosphatidylserine (PS) and phosphatidylinositol (PI). Under symmetrical KCl concentrations (3 apprx 1,000 mM), single channel conductances of maxi K channels in charged membranes were 1.1 apprx 1.7 times larger than those in PE membranes, and the differences were more pronounced at the lower ionic strength. The average slope conductances at 100 mM KCl were 251 +/- 9.9, 360 +/- 8.7 and 356 +/- 12.4 (mean +/- SEM) pS in PE, PS and PI membranes respectively. The potentials at which Po was 1/2, appeared to have shifted left by 40 mV along voltage axis in the membranes formed with PS or PI. Such shift was consistently seen at pCa 5, 4.5, 4 and 3.5. Estimation of the effect of surface charge from these data indicated that maxi K channels sensed the surface potentials at a distance of 8 apprx 9 ANG from the membrane surface. In addition, similar insulation distance (7 apprx 9 ANG) of channel mouth from the bilayer surface charge was predicted by a 3-barrier-2-site model of energy profile for the permeation of K+ ions. In conclusion, despite the differences in structure and fluidity of phospholipids in bilayers, the activities of maxi K channels in two charged membranes composed of PS or PI were strikingly similar and larger than those in bilayers of PE. These results suggest that the enhancement of conductance and Po of maxi channels is mostly due to negative charges in the phospholipid head groups.

Dual action of d-tubocurarine on large-conductance Ca2+-activated K+ channels from rat brain reconstituted into planar lipid bilayer

Using the planar lipid bilayer method, we investigated the effect of d-tubocurarine (dTC) on the extracellular side of large-conductance Ca2+-activated K+ channel from rat brain. When the initial open probability (Po) of the channel was relatively high, dTC decreased channel activity in a concentration dependent manner. In contrast, when the initial Po was lower, sub-micro molar dTC increased channel activity by destabilizing the closed states of the channel. Further addition of dTC up to micro molar range decreased channel activity. This dual effect of dTC implicates that there exist at least two different binding sites for dTC.

Fast and slow gating types of SR ryanodine receptor/channel purified from canine latissimus dorsi muscle

The ryanodine receptor/channel (RyR) mediates the release of calcium from the sarcoplasmic reticulum (SR) in both skeletal and cardiac muscle cells. There are three isoforms of the RyR: RyR1, RyR2, and RyR3. RyR1 is specifically expressed in skeletal muscles and RyR2 in cardiac muscles. RyR3 is yet another isoform found in non-muscle cells such as neuronal cells. Single channel recordings of RyR1 and RyR2 reconstituted in artificial lipid bilayer show that the characteristics of two isoforms are very distinct. RyR1 has a shorter mean open time and is activated at a higher concentration of Ca2+ than RyR2. In this study, we isolated the heavy SR membranes from canine latissimus dorsi muscles and investigated the single channel activities from the heavy SR membrane fraction using Cs+ as a charge carrier. Two different types of activities were observed. The fast-gating type (FG) with the mean open time of 0.9 ms was more frequently recorded (n = 12) than the slow-gating type (SG) with the mean open time of 269.2 ms. From the I-V relation, the slope conductance of the FG was calculated to be 514.7 pS and the SG, to 625.6 pS. The activity of the fast gating type increased by raising the concentration of Ca2+ in the cis-solution up to 100 microM. The appearance of the SG in the canine heavy SR membrane fraction suggests a possibility that two types of RyR isoform are co-expressed in mammalian skeletal muscle as well as in avian, amphibian and piscine fast twitch muscles.