The preservation of liposomes by raffinose family oligosaccharides during drying is mediated by effects on fusion and lipid phase transitions

Raffinose family oligosaccharides (RFO) have been implicated as protective agents in the cellular dehydration tolerance, especially of many plant seeds. However, their efficacy in stabilizing membranes during dehydration has never been systematically investigated. We have analyzed the effects of sucrose, raffinose, stachyose, and verbascose on liposome stability during air-drying. With increasing degree of polymerization (DP), the RFO were progressively better able to stabilize liposomes against leakage of aqueous content and against membrane fusion after rehydration. Indeed, there was a very tight linear correlation between fusion and leakage for all RFO. These data indicate that increased protection of liposomes against leakage with increasing DP is due to better protection against fusion. This is in accord with the higher glass transition temperature of the longer chain oligosaccharides. Further evidence for the influence of glass transitions on membrane stability in the dry state was provided by experiments testing the temperature dependence of membrane fusion. During incubation at temperatures up to 95 °C for 2 h, fusion increased less with temperature in the presence of higher DP sugars. This indicates that RFO with a higher glass transition temperature are better able to protect dry membranes at elevated temperatures. In addition, Fourier-transform infrared (FTIR) spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry liposomes in the presence of all investigated sugars. However, the RFO became slightly less effective with increasing chain length, again pointing to a decisive role for preventing fusion. A direct interaction of the RFO with the lipids was indicated by a strong effect of the sugars on the phosphate asymmetric stretch region of the infrared spectrum.     

Specific effects of fructo- and gluco-oligosaccharides in the preservation of liposomes during drying

The fructan family of oligo- and polysaccharides is a group of molecules that have long been implicated as protective agents in the drought and freezing tolerance of many plant species. However, it has been unclear whether fructans have properties that make them better protectants for cellular structures than other sugars. We compared the effects of fructans and glucans on membrane stability during air-drying. Although glucans of increasing chain length were progressively less able to stabilize liposomes against leakage of aqueous content after rehydration, fructans showed increased protection. On the other hand, glucans became more effective in protecting liposomes against membrane fusion with increasing chain length, whereas fructans became less effective. Fourier transform infrared spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature (T(m)) of air-dried liposomes by approximately 25 degrees C in the presence of sucrose and maltose. For the respective pentasaccharides, the reduction of T(m) of the lipids was 9 degrees C lower for samples containing fructan than for those containing glucan, indicating increased sugar--membrane interactions for the fructan compared to the glucan. A reduced interaction of the longer-chain glucans and an increased interaction of the respective fructans with the phospholipid head groups in the dry state was also indicated by dramatic differences in the phosphate asymmetric stretch region of the infrared spectrum. Collectively, our data indicate that the fructo-oligosaccharides accumulated in many plant species under stress conditions could indeed play an important role in cellular dehydration tolerance.     

Antioxidant activity of probucol and its effects on phase transitions in phosphatidylcholine liposomes

The effect of probucol on the phase behavior of dimyristoylphosphatidylcholine (DMPC) was examined by fluorescence polarization and differential scanning calorimetry (DSC). Probucol broadens and shifts the temperature of the main phase transition of DMPC liposomes as measured by fluorescence polarization with diphenylhexatriene and trimethyl-ammonium-diphenylhexatrine at concentrations as low as 5 mole%. As measured by DSC, probucol reduces the transition temperature of the gel----liquid-crystalline phase transition of DMPC by approx. 2 C degrees at all concentrations above about 5 mole% probucol and eliminates the pretransition at less than 1 mole%. In addition, the phase transition of DMPC is broadened and the enthalpy of the transition reduced by approx. 50%. Even at high concentrations of probucol, the gel----liquid-crystalline phase transition of DMPC is not eliminated. Similar effects are observed with dipalmitoylphosphatidylcholine liposomes. Based on these DSC measurements, measurements of the melting of probucol in dry mixtures with DMPC and observations of probucol mixtures with DMPC under polarizing optics, the maximum solubility of probucol in DMPC is approx. 10 mole%. This concentration exceeds that required (approx. 0.5 mole%) to prevent peroxidation of 10 mole% arachidonic acid in DMPC liposomes for 30 min in the presence of 0.05 mM Fe(NH4)(SO4)2 at 4 degrees C. Thus, probucol has a limited solubility in saturated phosphatidylcholine bilayers, but is an effective antioxidant at concentrations lower than its maximum solubility.     

myo-Inositol and sucrose concentrations affect the accumulation of raffinose family oligosaccharides in seeds

Raffinose family oligosaccharides (RFOs) fulfil multiple functions in plants. In seeds, they possibly protect cellular structures during desiccation and constitute carbon reserves for early germination. Their biosynthesis proceeds by the transfer of galactose units from galactinol to sucrose. Galactinol synthase (GolS), which mediates the synthesis of galactinol from myo-inositol and UDP-galactose, has been proposed to be the key enzyme of the pathway. However, no significant relationship was detected between the extractable GolS activity and the amount of RFOs in seeds from seven pea (Pisum sativum L.) genotypes selected for high variation in RFO content. Instead, a highly significant correlation was found between the levels of myo-inositol and RFOs. Moderately strong relationships were also found between sucrose and RFO content as well as between myo-inositol and galactinol. Further evidence for a key role of myo-inositol for the synthesis of galactinol was obtained by feeding exogenous myo-inositol to intact pea seeds and by the analysis of four barley (Hordeum vulgare L.) low phytic acid mutants. In seeds of three of these mutants, the reduced demand for myo-inositol for the synthesis of phytic acid (myo-inositol 1,2,3,4,5,6-hexakisphosphate) was associated with an increased level in myo-inositol. The mutants seeds also contained more galactinol than wild-type seeds. The results suggest that the extent of RFO accumulation is controlled by the levels of the initial substrates, myo-inositol and sucrose, rather than by GolS activity alone.     

The metabolism of galactose and the raffinose oligosaccharides by germinating bambarra groundnut seeds

Stachyose is present in the highest amount in the soluble sugar fraction of dry bambarra groundnut cotyledons, followed in descending order by raffinose, sucrose and verbascose. During germination in the dark, the stachyose and raffinose content decrease rapidly, but there is little change in the relatively small amount of verbascose present. The sucrose content increases rapidly during the first two weeks and decreases thereafter. Free glucose and fructose were present in the cotyledons after the 7th day and gradually increased in amount with time of germination. Free galactose and other galactose-containing oligosaccharides were not detected in either the dry or germinated bambarra seeds. During germination, galactose was the only identifiable sugar, aside from traces of sucrose, glucose and fructose, in the extracted soluble sugar fraction in the embryonic axes of all ages when the tissue was incubated with D-[1¹⁴C] galactose. With the cotyledons, however, most of the radioactivity was in glucose and fructose during the early period of germination and in sucrose later. A small fraction of the radioactivity was lost as CO₂.     

The effect of arbutin on membrane integrity during drying is mediated by stabilization of the lamellar phase in the presence of nonbilayer-forming lipids.

Arbutin (4-hydroxyphenyl-beta-glucopyranoside) is a solute accumulated to high concentrations in drought and frost resistant plants. Arbutin can inhibit membrane lysis, both free radical-mediated and enzymatic in nature, and it has been suggested that arbutin might contribute to membrane stabilization in these plants. However, we found that arbutin destabilized phosphatidylcholine vesicles during drying and rehydration, which appears to be inconsistent with the proposed protective function of arbutin for membranes. We also found, however, that arbutin stabilizes membranes containing nonbilayer-forming lipids during freezing. We now report that, in liposomes containing the nonbilayer-forming lipids monogalactosyldiacylglycerol (MGDG) or phosphatidylethanolamine (PE), arbutin served a protective function during drying, as measured by retention of carboxyfluorescein (CF) and extent of vesicle fusion. In hydrated samples containing these lipids, arbutin stabilized the lamellar liquid crystalline phase. Therefore, the interaction between arbutin and lipid membranes and the resulting effects on membrane stability depend, in a complex manner, on the lipid composition of the membrane.     

"Blocking" by polyethyleneglycol of single lipid pores arising in unmodified bilayer lipid membranes during phase transitions

Changes in the ionic permeability of bilayer lipid membranes from dipalmitoyl phosphatidylcholine at temperatures of phase transition in LiCl (1 M) solution after the addition of polyethyleneglycols of different molecular masses have been studied. The transition of ionic membrane channels from the conducting state to a blocking nonconducting state using polymers makes it possible to calibrate lipid pores. It was shown that low-molecular-weight glycerol, polyethyleneglycols with molecular masses of 300 and 600 decrease the amplitude of fluctuations of the current through the membrane, the decrease being proportional to the size of the polymer molecule being incorporated. The addition of polyethyleneglycols with molecular masses of 1450, 2000, and 3350 decrease the current fluctuations to the basal noise level. This result is considered as a complete blockade of ion channel conductivity. In the presence of rather large polymers, such as polyethyleneglycols with molecular masses of 6000 and 20000, which are practically not incorporated into the pore, single current fluctuations occur again; however, their amplitudes are somewhat smaller than in the absence of polyethyleneglycol. It is assumed that the complete blockade of the conductivity of lipid ionic channels by polyethyleneglycols with molecular masses of 1450, 2000, and 3350 is due to the dehydration of the pore gap and the conversion of the hydrophilic pore to a hydrophobic pore.     

Effect of melittin on thermotropic lipid state transitions in phosphatidylcholine liposomes.

We have examined the Raman scattering due to CH stretching vibrations, as well as to v(-C=C-) and v(=C-C=) of beta-carotene, of liposomes composed of phosphatidylcholine (egg, dimyristoyl, dipalmitoyl) +/- cholesterol, beta-carotene or melittin in the temperature range of -10 degrees C to 45 degrees C. (2) Plots vs. temperature of the intensities of the 2885 cm-1 and 2930 cm-1 CH stretching bands relative to the intensity of the thermally stable 2850 cm-1 band, i.e. the I2885/I2850 and I2930/I2850 ratios, reveal a sharp discontinuity in cholesterol-free phosphatidylcholine liposomes; this coincides with the gel leads to liquid-crystal transition temperature of the fatty acyl chains. In cholesterol/phosphatudylcholine liposomes the change in I2885/I2850 occurs over a very broad temperature range and I2930/I2850 remains stable. (3) I1527/I1158, i.e. the intensity of v(-C=C-) relative to that of v(=C-C-) in beta-carotene/phosphatidylcholine liposomes, changes discontinuously at the gel leads to liquid-crystal transition temperature. The values above the transition temperature approach those of the carotenoid in organic solution. (4) The transitions reported in I2885/I2850 for phosphatidylcholine/melittin liposomes (25-56; 1, M/M) are shifted to much higher temperatures than observed in phosphatidylcholine liposomes. In the case of dimyristoyl phosphatidylcholine/melittin the changes in I2930/I2850 also occurs at a higher temperature (28 degrees C) than without melittin (21 degrees C), but the temperature shift is less than the +13 degrees C observed for I2885/I2850. It appears that the apolar moiety of melittin organizes phospholipids adjacent to and more remote from the peptide moiety, to form complexes with an elevated lipid transition temperature. The effect of the peptide moiety is greater on the methylene segments (I2885/I2850) than on the methyl termini (I2930/I2850).     

Radioprotective effects of lipid A, liposomes, and liposomes containing lipid A in mice

Lipid A from Gram-negative bacterial lipopolysaccharide (endotoxin) was incorporated into liposomal membranes and examined as a prophylactic radioprotectant compound in lethally irradiated mice. Splenic hematopoietic activity, resulting in increased numbers of spleen cell colonies, was induced both by lipid A alone or more strongly by liposomal lipid A. Increased survival of lethally irradiated animals was induced to a slight extent by liposomes alone, to a greater extent by lipid A, and at the highest level by liposomes containing lipid A. Under conditions where 100% of untreated or saline-treated animals died of acute radiation syndrome after 20 days, more than 90% of the animals pretreated with liposomal lipid A were still alive 30 days after irradiation. We conclude that lipid A had substantial radioprotectant activity by itself, and the activity was enhanced by incorporation into liposomes. Liposomes alone also exhibited mild radioprotectant effects.     

The effects of lipid phase transitions on the interaction of mitochondrial NADH--ubiquinone oxidoreductase with ubiquinol--cytochrome c oxidoreductase.

1. The endogenous phosphatidylcholine and phosphatidylethanolamine of Complexes I and III from bovine heart mitochondria may be completely replaced with 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine with at least partial retention of activity. 2. The lipid-replaced enzymes associate in 1:1 molar ratio to give a Complex I--III unit catalysing NADH-cytochrome c oxidoreductase activity. 3. On increasing the concentration of ubiquinone-10 and the synthetic phospholipid, the lipid-replaced Complexes appear to operate independently of each other as in the natural membrane. Thus the lipid-replaced enzymes associate in exactly the same ways as the enzymes containing natural phospholipids. 4. Arrhenius plots of NADH--cytochrome c oxidoreductase activity reconstituted from lipid-replaced Complexes I and III exhibit changes in slope at 24 degrees C. When the concentrations of phospholipid and ubiquinone-10 are increased, the Arrhenius plots show discontinuities at 24 degrees C as well as changes in slope. 5. The kinetics of cytochrome b reduction by NADH were measured in mixtures containing 2 mol of Complex III/mol of Complex I. When the enzymes contained natural phospholipids. the reduction kinetics were biphasic. When the enzymes had been supplemented with further phospholipid and ubiquinone-10 the kinetics were monophasic. When lipid-replaced enzymes were supplemented with 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine and ubiquinone-10, reduction of cytochrome b was monophasic above the phase-transition temperature of the lipid but biphasic below it. 6. These findings are interpreted in terms of the model for the interaction of Complexes in the natural membrane proposed by Heron, Ragan & Trum-power [(1978) Biochem. J. 174, 791--800].     

Effect of lipid composition on HVJ-mediated fusion of glycophorin liposomes to erythrocytes

We previously reported that liposomes containing glycophorin or gangliosides, both of which were isolated from human erythrocytes, are efficiently fused to erythrocyte membranes in the presence of HVJ (Umeda, M. et al., J. Biochem. 94, 1955-1966 (1983), and Virology 133, 172-182 (1984]. In the present work, the effect of lipid composition in glycophorin liposomes on their sensitivity to fusion with erythrocytes was studied. Very little fusion occurred when glycophorin liposomes composed of dipalmitoylphosphatidylcholine-dicetylphosphate (9:1), dimyristoylphosphatidylcholine-dicetylphosphate (9:1), or egg yolk phosphatidylcholine-dicetylphosphate (9:1) were incubated with human erythrocytes in the presence of HVJ at 37 degrees C. Addition of cholesterol into these liposomal membranes greatly enhanced the sensitivity of the liposomes to fusion. The presence of phosphatidic acid and phosphatidylethanolamine in liposomes also enhanced the sensitivity, whereas the presence of lysophosphatidylcholine had no significant effect on the ability of the liposomes to fuse. The fusion efficiency of liposomes was also enhanced by the presence of glucosylceramide. Change of lipid composition in liposomes had, however, no appreciable influence on the HVJ-mediated binding of liposomes to erythrocytes, suggesting that the interaction between HANA protein of HVJ and glycophorin in liposomes was not affected by the lipid composition of the liposomes.     

Effect of delta-lysin on the phase transitions of lipid assemblies

X-ray small-angle diffraction, differential scanning calorimetry (DSC), and temperature scanning densitometry (TSD) were used to study the effect of -lysin on the phase transitions of lipid assemblies from 1,2-0-dixehadecyl-sn-glycero-3-phosphoholine (DHPC). The experiments were carried out in excess of water in a temperature range of 0–55 °C, and at low peptide concentrations between 10^-4 and 10^-2 moles peptide per mole phospholipid. The incorporation of -lysin into lipid assemblies alters the lipid structure without significant changes on the temperatures of phase transition from gel to liquid crystalline phase. The temperature of the main transition was nearly unaffected. A reduction in the transition volume of the lipids with increasing concentrations of -lysin was observed. The minor changes in these parameters were interpreted as long-range structural changes caused by the peptide incorporation. The results are discussed in terms of the concept of cooperative phase transition of entire clusters occurring within a membrane implying that relative stable domains of gel phase, and liquid crystalline phase co-exist.