Masataka Kusube

Pressure study on symmetric and asymmetric phospholipid bilayers: effect of vesicle size on Prodan fluorescence

The bilayer phase behavior of symmetric and asymmetric phosphatidylcholines (PCs), 1,2‐diheptadecanoyl‐PC (C17PC), 1‐palmitoyl‐2‐stearoyl‐PC (PSPC), and 1‐stearoyl‐2‐palmitoyl‐PC (SPPC), with different vesicle sizes were investigated by a high‐pressure fluorescence method using the polarity‐sensitive fluorescent probe Prodan. The second derivative of fluorescence spectra for all the PCs of small‐sized vesicle showed four minima characteristic of four membrane states on the spectra irrespective of the acyl‐chain symmetry, whereas those of large‐sized vesicle had one more minimum originating from the most hydrophilic site at the membrane surface. These findings indicate that Prodan molecules can distribute into multiple sites in the bilayer and move around the head‐group region depending on the vesicle size. The behavior of the spectra in the SPPC bilayer suggested that the interdigitated gel phase had a less polar “pocket” formed by a space between uneven terminal methyl ends of the sn‐1 and sn‐2 chains. It turned out that the curvature of vesicles affects the distribution of the Prodan molecules in all phases, more particularly in the interdigitated gel phase.

Barotropic and Thermotropic Phase Behavior of Bilayer Membranes Composed of Phospholipids Containing Cis Double Bond in the sn -2 Acyl Chain

The phase transitions of two kinds of saturated (in the sn-1 position) and unsaturated (in the sn-2 position) mixed-chain phospholipids, 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), were observed by high-pressure optical method and differential scanning calorimetry. The temperature-pressure phase diagrams were constructed on the basis of the obtained phase-transition data and the results were compared with those of saturated-chain phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and those of unsaturated-chain phospholipids, 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC). The phase transition temperatures of mixed-chain lipids showed the intermediate values for the bilayer membranes between saturated-and unsaturated-chain lipids. The thermodynamic quantities of the transition for mixed- and unsaturated-chain lipid bilayer membranes were smaller than those for saturated-chain lipids. The transition temperatures of POPE and POPC bilayer membranes were significantly different although the thermodynamic quantities of the transition for both bilayers resembled each other in magnitude.

Effects of Cis and Trans Unsaturation on the Barotropic and Thermotropic Phase Behavior of Phospholipid Bilayers

In order to understand the effect of trans and cis unsaturation on the thermotropic and barotropic phase transition of lipid bilayer membranes, the phase transitions of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and the corresponding phosphocholines (i. e., DSPC, DEPC and DOPC, respectively) were observed by high-pressure optical method and differential scanning calorimetry. For all of the lipid bilayers studied, the temperatures of transition from the gel phase to the liquid crystalline phase were almost linearly elevated by pressure. The substitution of trans or cis unsaturated acyl chain for the saturated acyl chain brought about the depression of phase-transition temperature. The values of ΔH and ΔV for the transition were remarkably reduced by the introduction of trans or cis double bond into the acyl chains. This situation is attributable to the geometrical configurations of cis and trans double bonds, which brings about the random packing of acyl chains in the gel state.

Effect of pressure on the bilayer phase transitions of N-methylated dipalmitoylphosphatidylethanolamines

The main-transition temperatures between gel and liquid crystalline phases for dipalmitoylphosphatidylmonomethylethanolamine (DPMePE) and dipalmitoylphosphatidyldimethylethanolamine (DPMe2PE) under high pressure were determined from differential scanning calorimetry (DSC) and optical methods. The thermodynamic quantities of main transition (enthalpy, entropy, and volume) were calculated and the results were compared with those of dipalmitoylphosphatidylethanolamine (DPPE) and dipalmitoylphosphatidylcholine (DPPC) to clarify the effect of N-methylation of DPPE on the main transition. The main-transition temperatures of N-methylated DPPEs increased linearly by applying pressure while they decreased with increasing the size of head group by stepwise N-methylation. On the other hand, there was no significant difference in thermodynamic quantities between the phospholipids. The difference in hydrophilic interactions between the different-sized polar head groups produces a great difference on the main-transition temperature and the packing arrangement of bilayer membrane in the gel phase.

Recovery of tobacco BY-2 cells after high hydrostatic pressure treatment

The recovery of Nicotiana tabacum L. cv. Bright Yellow 2 (BY‐2) cells in Linsmaire and Skoog medium after treatment at high hydrostatic pressure was investigated using an Evans Blue staining method to discriminate live from dead cells. The survival of BY‐2 cells just after the high‐pressure treatment at 5° C and 25° C decreased abruptly at pressures higher than 50 MPa and 100 MPa, respectively. Furthermore, almost all of the BY‐2 cells treated at 5° C and 25° C recovered pressures below 25 MPa and 75 MPa, respectively. However, no BY‐2 cells recovered at pressures above 100 MPa at either temperature.