Direct Comparison of Disaccharide Interaction with Lipid Membranes at Reduced Hydrations.

Understanding sugar-lipid interactions during desiccation and freezing is an important step in the elucidation of cryo- and anhydro-protection mechanisms. We determine sucrose, trehalose, and water concentration distributions in intra-bilayer volumes between opposing dioleoylphosphatidylcholine bilayers over a range of reduced hydrations and sugar concentrations. Stacked lipid bilayers at reduced hydration provide a suitable system to mimic environmental dehydration effects, as well as a suitable system for direct probing of sugar locations by neutron membrane diffraction. Sugar distributions show that sucrose and trehalose both behave as typical uncharged solutes, largely excluded from the lipid bilayers regardless of sugar identity, and with no correlation between sugar distribution and the lipid headgroup position as the hydration is changed. These results are discussed in terms of current opinions about cryo- and anhydro-protection mechanisms.

Development of cryopreservation for Loxocarya cinerea---an endemic Australian plant species important for post-mining restoration.

We report the development of a cryopreservation protocol for the endemic Western Australian plant species Loxocarya cinerea (Restionaceae). Shoot tips from two genotypes, SXH404 and SXH804, were cryopreserved using the droplet-vitrification technique. Control explants, which were cryoprotected, but not cooled, showed regeneration for both genotypes (SXH404, 22.1 +/- 5.9%; SXH804, 67.7 +/- 9.6%). Extension of incubation in PVS2 from 30 to 60 min did not lead to survival after cryopreservation. Thermal analysis using differential scanning calorimetry confirmed the beneficial effect of a loading phase but also revealed no or very little ice formation after cryoprotection of shoot tips in other treatments. Regeneration following cryopreservation was obtained for genotype SXH804 (4.3 +/- 2.1%) but not for SXH404. Regenerated explants of L. cinerea SXH804 were morphologically identical to tissue-cultured plants. As an alternative to shoot tips, callus tissues of clone SXH404 were successfully cryopreserved (> 66.7% post LN survival) using the same protocol.

Phase separation in a ternary DPPC/DOPC/POPC system with reducing hydration.

The maintenance of plasma membrane structure is vital for the viability of cells. Disruption of this structure can lead to cell death. One important example is the macroscopic phase separation observed during dehydration associated with desiccation and freezing, often leading to loss of permeability and cell death. It has previously been shown that the hybrid lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) can act as a line-active component in ternary lipid systems, inhibiting macroscopic phase separation and stabilising membrane microdomains in lipid vesicles [1]. The domain size is found to decrease with increasing POPC concentration until complete mixing is observed. However, no such studies have been carried out at reduced hydration. To examine if this phase separation is unique to vesicles in excess water, we have conducted studies on several binary and ternary model membrane systems at both reduced hydration ("powder" type samples and oriented membrane stacks) and in excess water (supported lipid bilayers) at 0.2 mol fraction POPC, in the range where microdomain stabilisation is reported. Differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR) are used to map phase transition temperatures, with X-ray and neutron scattering providing details of the changes in lipid packing and phase information within these boundaries. Atomic force microscopy (AFM) is used to image bilayers on a substrate in excess water. In all cases, macroscopic phase separation was observed rather than microdomain formation at this molar ratio. Thus POPC does not stabilise microdomains under these conditions, regardless of the type of model membrane, hydration or temperature. Thus we conclude that the driving force for separation under these conditions overcomes any linactant effects of the hybrid lipid.

Localization of trehalose in partially hydrated DOPC bilayers: insights into cryoprotective mechanisms.

Trehalose, a natural disaccharide with bioprotective properties, is widely recognized for its ability to preserve biological membranes during freezing and dehydration events. Despite debate over the molecular mechanisms by which this is achieved, and that different mechanisms imply quite different distributions of trehalose molecules with respect to the bilayer, there are no direct experimental data describing the location of trehalose within lipid bilayer membrane systems during dehydration. Here, we use neutron membrane diffraction to conclusively show that the trehalose distribution in a dioleoylphosphatidylcholine (DOPC) system follows a Gaussian profile centred in the water layer between bilayers. The absence of any preference for localizing near the lipid headgroups of the bilayers indicates that the bioprotective effects of trehalose at physiologically relevant concentrations are the result of non-specific mechanisms that do not rely on direct interactions with the lipid headgroups.