Membrane and protein properties of freeze-dried mouse platelets

Membrane properties and the overall protein secondary structure of freeze-dried trehalose-loaded mouse platelets were studied using steady state fluorescence anisotropy and Fourier transform infrared spectroscopy (FTIR). FTIR results showed that fresh control mouse platelets have a main phase transition at approximately 14 degrees C, whereas, freeze-dried platelets exhibited a main phase transition approximately 12 degrees C. However, the cooperativity of the transition of the rehydrated platelets was greatly enhanced compared to that of control platelets. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed that the lipid order in the core of the membrane was affected by freeze-drying procedures. Similar experiments with trimethyl ammonium 1,6 diphenyl-1,3,5 hexatriene (TMA-DPH), a membrane surface probe, indicated that membrane properties at the membrane/water interface were less affected by freeze-drying procedures than the core of the membrane. Lyophilization did not result in massive protein denaturation, but the overall protein secondary structure was altered, based on in situ assessment of the amide-I and amide-II band profiles. Lyophilization-induced changes to endogenous platelet proteins were further investigated by studying the protein's heat stability. In fresh control platelets, proteins denatured at 42 degrees C, whereas proteins in the rehydrated platelets denatured at 48 degrees C.     

Temperature dependence of fluid phase endocytosis coincides with membrane properties of pig platelets

In previous studies we have shown that platelets take up low molecular weight molecules from the medium by fluid phase endocytosis, a phenomenon that we previously have used to load trehalose into human platelets, after which we have successfully freeze-dried them. We now extend those findings to a species to be used in animal trials of freeze-dried platelets:pigs. Further, we report results of studies aimed at elucidating the mechanism of the uptake. Temperature dependence of fluid-phase endocytosis was determined in pig platelets, using lucifer yellow carbohydrazide (LY) as a marker. A biphasic curve of marker uptake versus temperature was obtained. The activation energy was significantly higher above 22 degrees C (18.7+/-1.8 kcal/mol) than below that critical temperature (7.5+/-1.5 kcal/mol). The activation energy of fluid phase endocytosis in human platelets was 24.1+/-1.6 kcal/mol above 15 degrees C. In order to establish a correlation between the effect of temperature on fluid phase endocytosis and the membrane physical state, Fourier transform infrared spectroscopy (FTIR) and fluorescence anisotropy experiments were conducted. FTIR studies showed that pig platelets exhibit a main membrane phase transition at approximately 12 degrees C, and two smaller transitions at 26 and 37 degrees C. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed a major transition at 8 degrees C and smaller transitions at 26 and 35 degrees C. In order to investigate the relative roles of known participants in fluid phase endocytosis, the effects of several chemical inhibitors were investigated. LY uptake was unaffected by colchicine, methylamine, and amiloride. However, disruption of specific microdomains in the membrane (rafts) by methyl-beta-cyclodextrin reduced uptake of LY by 35%. Treatment with cytochalasin B, which inhibits actin polymerization, reduced the uptake by 25%. We conclude that the inflection point in the LY uptake versus temperature plot at around 22 degrees C is correlated with changes in membrane physical state, and that optimal LY internalization requires an intact cytoskeleton and intact membrane rafts.     

Temperature dependence of fluid phase endocytosis coincides with membrane properties of pig platelets

In previous studies we have shown that platelets take up low molecular weight molecules from the medium by fluid phase endocytosis, a phenomenon that we previously have used to load trehalose into human platelets, after which we have successfully freeze-dried them. We now extend those findings to a species to be used in animal trials of freeze-dried platelets:pigs. Further, we report results of studies aimed at elucidating the mechanism of the uptake. Temperature dependence of fluid-phase endocytosis was determined in pig platelets, using lucifer yellow carbohydrazide (LY) as a marker. A biphasic curve of marker uptake versus temperature was obtained. The activation energy was significantly higher above 22 °C (18.7±1.8 kcal/mol) than below that critical temperature (7.5±1.5 kcal/mol). The activation energy of fluid phase endocytosis in human platelets was 24.1±1.6 kcal/mol above 15 °C. In order to establish a correlation between the effect of temperature on fluid phase endocytosis and the membrane physical state, Fourier transform infrared spectroscopy (FTIR) and fluorescence anisotropy experiments were conducted. FTIR studies showed that pig platelets exhibit a main membrane phase transition at approximately 12 °C, and two smaller transitions at 26 and 37 °C. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed a major transition at 8 °C and smaller transitions at 26 and 35 °C. In order to investigate the relative roles of known participants in fluid phase endocytosis, the effects of several chemical inhibitors were investigated. LY uptake was unaffected by colchicine, methylamine, and amiloride. However, disruption of specific microdomains in the membrane (rafts) by methyl-β-cyclodextrin reduced uptake of LY by 35%. Treatment with cytochalasin B, which inhibits actin polymerization, reduced the uptake by 25%. We conclude that the inflection point in the LY uptake versus temperature plot at around 22 °C is correlated with changes in membrane physical state, and that optimal LY internalization requires an intact cytoskeleton and intact membrane rafts.     

Lipid peroxidation causes an increase of lipid order and a decrease of 5'-nucleotidase activity in the liver plasma membrane.

The effect of peroxidation on 5'-nucleotidase activity as well as on membrane microviscosity has been investigated in liver plasma membranes from Wistar rats. The peroxidation was performed with 100 microM H2O2 and 200 microM FeSO4 and/or with 5 mM t-butylhydroperoxide. Treatment of the membranes with these oxidizing agents resulted in an elevation of the transition temperatures of the polarization of the lipid fluorescent probes 1,6 diphenyl-1,3,5 hexatriene (DPH), 3-p-(6-phenyl) 1,3,5 hexatriene phenylpropionic acid (PA-DPH) as well as of the fluorescent thiol reagent N-(1-pyrene) maleimide (1-PM). The peroxidation resulted in a decrease of the activity of 5'nucleotidase. Our data support that the increase of membrane microviscosity of the lipid domain regulates the activity of 5'-nucleotidase.     

The Structure of Destruxin B,a Toxic Metabolite of Oospora destructor

The viability of freeze-dried Lactobacillus bulgaricus B-1 was affected by rehydration temperature, and maximum recovery of the viable cells was obtained when they were rehydrated at 20 to 25°C. Cellular ribonucleotides leaked out from the freeze-dried cells during rehydration, but there was no correlation between the viability of cells and the amount of leaked substances. Rehydration of the freeze-dried cells in the presence of RNase caused marked loss of viability. These results suggest that the cell surface was damaged by freeze-drying and its selective permeability was lost to some extent.     

Temperature-induced transitions of porcine intestinal brush border membranes

Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33°C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35°C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35°C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35°C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33°C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.     

Effects of freeze-preservation on some pollen enzymes: II. Freezing and freeze-drying stresses

The isozymes of lily and corn pollen esterases and acid phosphatase were studied in relation to freeze-drying and vacuum-drying. Fresh samples of Lilium longiflorum L. and Zea mays L. pollen were frozen at rates ranging between 200 and 100 °C/min and freeze-dried at temperatures from 0 to ?70 °C for approximately 48 to 70 hr. Freeze-dried samples were rehydrated slowly (10% relative humidity) and rapidly (90% relative humidity). Vacuum-drying was performed at room temperature (22 °C).Soluble pollen enzymes were analyzed by disc electrophoresis on polyacrylamide gels stained with substrate specific reagents. The stained gels were evaluated by densitometry for migration rate, isozyme pattern, and relative activity. The numerical data generated in this manner were then statistically analyzed.The following conclusions resulted from this study: (i) freeze-drying and freeze-thawing treatments were comparable except for corn esterases; (ii) freeze-drying induced alterations in enzyme activity except for corn acid phosphatase; (iii) the freezing rate, the final freezing temperature, and exposure to various relative humidities produced few changes in freeze-dried material; (iv) freeze-drying was less detrimental than vacuum-drying to the enzyme characteristics of corn; (v) freeze-drying yielded higher viabilities than vacuum-drying; and (vi) acid phosphatase alterations appeared to be related to pollen viability in most cases.     

Lipid phase separation correlates with activation in platelets during chilling

When human platelets are chilled below 22°C, they spontaneously activate, a phenomenon that severely limits their storage life. It has previously been proposed that there is a correlation between cold-induced platelet activation and passage of the membranes through a liquid-crystalline to gel phase transition. Because animal models are essential for developing methods for cold storage of platelets, it is necessary to investigate such a correlation in animal platelets. In this work, horse platelets were used as a model, and it was found thatcoldinduced morphological activation is related to the lipid phase transition. Using fluorescence microscopy with the lipophilic fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-C₁₈), and Fourier transform infrared spectroscopy (FTIR), it was found that lipid phase separation occurs during cooling and low temperature storage. Furthermore, removal of cholesterol from the plasma membrane also induced a phase separation, possibly between specific phospholipid classes. Steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-DPH (TMA-DPH) were compared in cells and multilamellar vesicles (MLV)composed of platelet lipids. Cholesterol depletion led to a decrease in the fluorescence anisotropy of the two probes, which can be explained by changes in the order of the phospholipid molecules. In addition, the lipid composition and fatty acid profile of the cellular phospholipids were determined. Based ofthe similarities between horse and human platelets, it is suggested that horse platelets may be used as a model for studying cold-stored platelets. The results are discussed in relation to the possible role of phase separation during cell signalling.     

Human platelets loaded with trehalose survive freeze-drying.

Human blood platelets are stored in blood banks for 5 days, after which they are discarded, by federal regulation. This short lifetime has led to a chronic shortage of platelets, a problem that is particularly acute in immunosuppressed patients, such as those with AIDS. We report here that platelets can be preserved by freeze-drying them with trehalose, a sugar found at high concentrations in organisms that naturally survive drying. We suggest that these findings will obviate the storage problem with platelets. Trehalose is rapidly taken up by human platelets at 37 degrees C, with loading efficiencies of 50% or greater. Fluid-phase endocytosis plays an important role in this efficient uptake of trehalose, but other mechanisms may also be involved. Trehalose-loaded platelets were successfully freeze-dried, with excellent recovery of intact platelets. Rehydration from the vapor phase led to a survival rate of 85%. The response of these platelets to the agonists thrombin (1 U/ml), collagen (2 microg/ml), ADP (20 micromM), and ristocetin (1.6 mg/ml) was almost identical to that of fresh, control platelets. Analysis by Fourier transform infrared spectroscopy demonstrated that the membrane and protein components of trehalose-loaded platelets after freeze-drying, prehydration, and rehydration were remarkably similar to those of fresh platelets.     

Enhancement in the viability and biosensing activity of freeze-dried recombinant bioluminescent bacteria

The genetically-engineeredEscherichia coli strain, DPD2540, which contains afabA::luxCDABE fusion gene, gives a bioluminescent output when membrane fatty acid synthesis is needed. For more practical application of this strain in the field as biosensor, freeze-drying was adopted. A 12% sucrose solution with Luria-Bertani (LB) broth, as determined by the viability after freeze-drying, was found to be the most effective composition for lyophilization solution among various compositions tested. Rapid freezing with liquid nitrogen also gave the best viability after freeze-drying as compared to samples frozen at −70°C and −20°C. The biosensing activities of the cells showed a greater sensitivity when the cells from the exponential phase were freeze-dried. Finally, the optimum temperature for use of the freeze-dried cells in the biosensor field was determined.     

Influence of Phospholipid Species on Membrane Fluidity: A Meta-analysis for a Novel Phospholipid Fluidity Index

Generalized membrane lipid composition determinants of fluidity have been widely investigated, including phospholipid/cholesterol ratio and unsaturation index. Individual phospholipids differ in their physical characteristics, including their interaction with cholesterol and level of unsaturation, emphasizing the importance of examining their individual influence on membrane fluidity. Thus, the purpose of this study was to examine the dominant phospholipids of biological membranes (phosphatidylcholine, PC; phosphatidylethanolamine, PE; sphingomyelin, SM) through a meta-analysis to assess the validity of an inclusive phospholipid fluidity index (PFI = PC/(PE + SM)) as a determinant for membrane fluidity (expressed as polarization of fluorescent probe 1,6 diphenyl-1,3,5-hexatriene) in comparison to previous phospholipid ratios (PC/PE and PC/SM). The results demonstrate that all indices significantly predicted membrane fluidity at 25°C (based on 10–13 data points). In contrast, only PFI approached significance when predicting membrane fluidity at 37°C (P = 0.10 based on five points). As a result, PFI appears to be the only phospholipid index close to significantly predicting membrane fluidity at mammalian physiological temperature. Because this meta-analysis only assessed studies using mammalian membranes, future work should experimentally assess the validity of the PFI utilizing membranes from mammals and a variety of other species and tissues at their respective physiological temperatures.     

Stabilization of a recombinant human epidermal growth factor parenteral formulation through freeze-drying

Development studies were performed to design a pharmaceutical composition that allows the stabilization of a parenteral rhEGF formulation in a lyophilized dosage form. Unannealed and annealed drying protocols were tested for excipients screening. Freeze-dry microscopy was used as criterion for excipients and formulation selection; as well as to define freeze-drying parameters. Excipients screening were evaluated through their effect on freeze-drying recovery and dried product stability at 50 °C by using a comprehensive set of analytical techniques assessing the chemical stability, protein conformation and bioactivity. The highest stability of rhEGF during freeze-drying was achieved by the addition of sucrose or trehalose. After storing the dried product at 50 °C, the highest stability was achieved by the addition of dextran, sucrose, trehalose or raffinose. The selected formulation mixture of sucrose and dextran could prevent protein degradation during the freeze-drying and delivery processes. The degradation rate assessed by RP-HPLC could decrease 100 times at 37 °C and 70 times at 50 °C in dried with respect to aqueous formulation. These results indicate that the freeze-dried formulation represents an appropriate technical solution for stabilizing rhEGF.     

Selective inactivation of the infectivity of freeze-dried Sendai virus by heat

The infectivity of freeze-dried Sendai virus was destroyed after heating at 100 ° C for 20 min while the hemagglutinin (HA) titer and the hemolytic (HL) activity were not affected. The HA titer was unaltered after heating at up to 140 ° C for 30 min. The HL activity was increased after freeze-drying, further increased after heating of freeze-dried virus at 115 ° C for 20 min, but was destroyed after heating for 30 min at 140 ° C.The selective heat inactivation of freeze-dried Sendai virus could be of use in the production of myxovirus vaccines and inactivated virus for cell-fusion studies.     

Optimisation and comparison of liquid and dry formulations of the biocontrol yeast <Emphasis Type="Italic">Pichia anomala</Emphasis> J121

The biocontrol yeast Pichia anomala J121 can effectively reduce mould growth on moist cereal grains during airtight storage. Practical use of microorganisms requires formulated products that meet a number of criteria. In this study we compared different formulations of P. anomala. The best way to formulate P. anomala was freeze-drying. The initial viability was as high as 80%, with trehalose previously added to the yeast. Freeze-dried products could be stored at temperatures as high as 30 °C for a year, with only a minor decrease in viability. Vacuum-drying also resulted in products with high storage potential, but the products were not as easily rehydrated as freeze-dried samples. Upon desiccating the cells using fluidised-bed drying or as liquid formulations, a storage temperature of 10 °C was required to maintain viability. Dependent on the type of formulation, harvesting of cells at different nutritional stresses affected the initial viabilities, e.g. the initial viability for fluidised-bed-dried cells was higher when the culture was fed with excess glucose, but for freeze-drying it was superior when cells were harvested after depletion of carbon. Using micro-silos we found that the biocontrol activity remained intact after drying, storage and rehydration for all formulations.     

Competition between cis,trans and Cyclopropane Fatty Acid Formation and its Impact on Membrane Fluidity

The impact of cis, trans and cyclopropane fatty acids on membrane fluidity was investigated using batch‐grown Pseudomonas putida P8 and Comamonas testosteroni ATCC 17454. A major difference observed between the two investigated strains is the absence of the ability to synthesize trans‐unsaturated fatty acids in Comamonas. When grown exponentially at 30 °C, a shift to 35 °C increased the trans/cis ratios of the fatty acids of P. putida P8 from 0 to 0.81 and 0 to 1.07, in lipid extracts and cell hydrolyzates, respectively. After prolonged growth followed by nutrient deprivation for 48 h, both at 30 °C, trans fatty acids were absent, but the cyclo/cis ratios rose from 0.1 to 1.55 in lipid extracts, and from 0.1 to 1.54 in cell hydrolyzates. C. testosteroni ATCC 17454 contained no cyclo fatty acids when harvested in the exponential phase after 6 h, whereas after 72 h cultivation, the cyclo/cis ratios rose to 0.49 and 0.47, in lipid extracts and cell hydrolyzates, respectively. Trans fatty acids were never observed in this strain. Increased cyclo/cis and trans/cis ratios correlated with decreased fluidity measured by the fluorescence anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene (DPH) intercalated in the bilayers of liposomes and by Fourier Transform Infrared (FTIR) spectroscopy of lipids prepared from the cells. The specific effect of cyclopropane fatty acids on membrane fluidity was much smaller than that of trans fatty acids. FTIR‐measurements of intact cells of P. putida P8 confirmed the high potency of trans fatty acids to decrease the fluidity. In cells with induced cyclopropane fatty acid synthesis, the membranes remained more fluidized, indicating the lower importance of these fatty acids for homeoviscosis.     

Heparin inhibition and polyamine stimulation of a glycogen synthase kinase (PC0.7) from rabbit skeletal muscle

Molecular motion of 1,6-diphenyl-1,3,5-hexatriene embedded in intact guinea pig alveolar macrophage membranes was investigated by using techniques of nanosecond timeresolved fluorescence anisotropy measurements in the temperature range of 0–50 °C, and as a function of benzyl alcohol concentration. It was shown that molecular arrangement and microheterogeneity of the hydrocarbon region surrounding 1,6-diphenyl-1,3,5-hexatriene molecules are dependent on the temperature and benzyl alcohol concentration. The lipid orientation order parameter, S_v, showed a discontinuity in the temperature range 12–40 °C, which may indicate a phase transition. N-Formylmethionylphenylalanine-stimulated production of O₂^? from macrophages increased with temperature parallel with changes in S_v. Benzyl alcohol decreases the magnitude of the lipid order parameter at all temperatures studied. In the same concentration range of benzyl alcohol, stimulated O₂^? production by macrophages was inhibited. These data show the complex relationship between lipid integrity in macrophage membranes and a physiological function of these cells. In addition, the results indicate that benzyl alcohol influences the integrity of both the protein and lipid hydrophobic regions of the membrane.     

Age related changes in fluidity of rat renal brushborder membrane vesicles

Fluorescence anisotrophy of 1,6 diphenyl-1,3,5-hexatriene was determined in renal brushborder membranes prepared from rats 7, 14, 21 and 28 days of age, and adults, from 5 degrees C to 45 degrees C. There is a parallel relationship between temperature and mean fluorescence anisotrophy in the different age groups with a progressive decrease in fluidity with age. There is no phase transition apparent in membranes from any age group as evidenced by the lack of a fluorescence polarization "break point". There is also a linear relationship between limiting hindered anisotrophy and previously determined values for the height of the Na+-proline overshoot. This suggests that the physical characteristics of the renal brushborder membrane responsible for differences in fluidity are related to age-dependent transport alterations.     

Viable rat offspring derived from oocytes intracytoplasmically injected with freeze-dried sperm heads

A 2 x 3 factorial designed experiment was conducted in order to examine whether freeze-dried rat spermatozoa can participate in full-term development following intracytoplasmic sperm injection (ICSI). A sperm suspension from cauda epididymides of Sprague-Dawley (SD) rats was prepared with or without ultrasonic treatment. The sonicated and non-sonicated sperm suspensions were processed for freeze-thawing (FT groups; 100 microl sample was cooled in liquid nitrogen vapor, stored for 1 day at--196 degrees C, and thawed in a 25 degrees C water bath) and freeze-drying (FD groups; 100 microl sample was frozen in liquid nitrogen for 20 s, lyophilized for 6 h, stored at 4 degrees C for 2 days, and rehydrated with 100 microl ultrapure water), or were subjected to immediate use for ICSI (fresh control groups). The sperm heads were microinjected into denuded SD oocytes using a piezo-driven micropipette 2-4 microm in diameter. The presumptive zygotes were transferred into oviducts of pseudopregnant Wistar female rats. Viable rat offspring were produced from all six experimental groups. Ultrasonic treatment of rat spermatozoa was effective in increasing the offspring rate (23.3% vs 6.7% in fresh control groups, 35.0% vs 7.6% in FT groups, 9.2% vs 2.5% in FD groups). The acrosomal region appeared to be intact even after ultrasonic FT and FD treatments as well as in the fresh controls, while the lateral dorsal region of the sperm membrane was more or less damaged in the sonicated, FT and FD samples. Thus, the successful participation of freeze-dried spermatozoa in full-term development was demonstrated by applying ICSI in the rat.     

DPH lifetime distributions in vesicles containing phospholipid hydroperoxides

Multifrequency phase fluorometry was used to determine the lifetime distributions of 1,6 diphenyl-1,3,5-hexatriene in 1-palmitoyl-2 linoleoyl phosphatidylcholine small unilamellar vesicles containing 2% incorporation of phospholipid hydroperoxides. A biexponential decay was observed in both vesicle preparations over a temperature range of 5 to 35 degrees C. Vesicles containing phospholipid hydroperoxides showed an overall longer lifetime as well as a greater distribution in width. These findings suggest that phospholipid hydroperoxides create structural heterogeneity in membrane structure.     

The use of the fluorescent probe α-parinaric acid to determine the physical state of the intracytoplasmic membranes of the photosynthetic bacterium, Rhodopseudomonas sphaeroides

α-Parinaric acid has been used to determine the degree of ordering of the hydrocarbon region of purified intracytoplasmic membranes of Rhodopseudomonas sphaeroides. The usefulness of α-parinaric acid as a probe of membrane fluidity was established by comparison of its fluorescent properties in phosphatidylcholine vesicles with those of the more commonly used fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene. Both fluorescent probes were shown to monitor similar environments in the phosphatidylcholine vesicles when the phospholipids were maintained at temperatures above their phase transition temperature.The rotational mobility of α-parinaric acid in the intracytoplasmic membranes was determined from 0 to 50°C, a region where no phase transitions were detectable. The rotational mobility of α-parinaric acid dissolved in vesicles formed from total extracted intracytoplasmic membrane phospholipids, was 2–3-fold greater than that measured in the intact intracytoplasmic membranes; demonstrating that the presence of protein greatly reduces the mobility of the phospholipid acyl chains of the intracytoplasmic membranes. Due to the high protein content of these membranes, the perturbing effect of protein on acyl chain mobility may extend to virtually all the intracytoplasmic membrane phospholipid.