α-Lactalbumin binding and membrane integrity—effect of charge and degree of unsaturation of glycerophospholipids

Several studies have shown that the physical state of the phospholipid membrane has an important role in protein-membrane interactions, involving both electrostatic and hydrophobic forces. We have investigated the influence of the interaction of the calcium-depleted, (apo)-conformation of bovine α-lactalbumin (BLA) on the integrity of anionic glycerophospholipid vesicles by leakage experiments using fluorescence spectroscopy. The stability of the membranes was also studied by measuring surface tension/molecular area relationships with phospholipid monolayers. We show that the degree of unsaturation of the acyl chains and the proportion of charged phospholipid species in the membranes made of neutral and acidic glycerophospholipids are determinants for the association of BLA with liposomes and for the impermeability of the bilayer. Particularly, tighter packing counteracted interaction with BLA, while unsaturation—leading to looser packing—promoted interaction and leakage of contents. Equimolar mixtures of neutral and acidic glycerophospholipids were more permeable upon protein binding than pure acidic lipids. The effect of lipid structure on BLA-membrane interaction and bilayer integrity may throw new light on the membrane disrupting mechanism of a conformer of human α-lactalbumin (HAMLET) that induces death of tumour cells but not of normal cells.     

Biophysical properties of membrane lipids of anammox bacteria: I. Ladderane phospholipids form highly organized fluid membranes

Anammox bacteria that are capable of anaerobically oxidizing ammonium (anammox) with nitrite to nitrogen gas produce unique membrane phospholipids that comprise hydrocarbon chains with three or five linearly condensed cyclobutane rings. To gain insight into the biophysical properties of these ‘ladderane’ lipids, we have isolated a ladderane phosphatidylcholine and a mixed ladderane phosphatidylethanolamine/phosphatidylglycerol lipid fraction and reconstituted these lipids in different membrane environments. Langmuir monolayer experiments demonstrated that the purified ladderane phospholipids form fluid films with a relatively high lipid packing density. Fluid-like behavior was also observed for ladderane lipids in bilayer systems as monitored by cryo-electron microscopy on large unilamellar vesicles (LUVs) and epi-fluorescence microscopy on giant unilamellar vesicles (GUVs). Analysis of the LUVs by fluorescence depolarization revealed a relatively high acyl chain ordering in the hydrophobic region of the ladderane phospholipids. Micropipette aspiration experiments were applied to study the mechanical properties of ladderane containing lipid bilayers and showed a relatively high apparent area compressibility modulus for ladderane containing GUVs, thereby confirming the fluid and acyl chain ordered characteristics of these lipids. The biophysical findings in this study support the previous postulation that dense membranes in anammox cells protect these microbes against the highly toxic and volatile anammox metabolites.     

Viability, maturation and embryo development in vitro of immature porcine and ovine oocytes vitrified in different devices

This study was designed to evaluate the effects of vitrification on immature porcine and ovine oocytes, collected at a slaughterhouse, by performing vitrification in devices with different volumes. Viability was evaluated both before and after vitrification and maturation. Immediately after warming, the percentage of viable pig oocytes was 81% regardless the type of device, while in the control (after oocyte selection) was 95%. The viability of matured pig oocytes after warming, vitrified in beveled edge open straws (BES) was 6%, in small-open-pulled-straw (SOPS) was 17% and in cryotop was 4%, while the viability of the control group was 86%. The viability and maturation results were similar with all devices. Embryo development (ED) was observed in fresh porcine oocytes with 15% 2-8 cell embryos, 7% morulae and 3% blastocysts, and non-embryo cleavage was observed in warmed oocytes. The viability of sheep oocytes immediately after warming averaged 90% in all devices, while that of the control (after oocyte selection) averaged 95%. The viability of warmed oocytes after maturation was: BES 21%, SOPS 30%, cryotop 21% and control group 86%; while maturation values were 11, 21, 34 and 70%, respectively. After vitrification, the highest ED was achieved with ovine oocytes vitrified in SOPS, with 17% morulae development and it was the only device in which blastocysts developed. A direct relationship was observed between viability and actin filament integrity in both species.