Structure and mechanical properties of giant lipid (DMPC) vesicle bilayers from 20 degrees C below to 10 degrees C above the liquid crystal-crystalline phase transition at 24 degrees C

We have used micromechanical tests to measure the thermoelastic properties of the liquid and gel phases of dimyristoylphosphatidylcholine (DMPC). We have found that the rippled P beta' phase is only formed when a vesicle is cooled to temperatures below the main acyl chain crystallization transition, Tc, under zero or very low membrane tension. We also found that the P beta' surface ripple or superlattice can be pulled flat under high membrane tension into a planar structure. For a ripple structure formed by acyl chains perpendicular to the projected plane, the projected area change that results from a flattening process is a direct measure of the molecular crystal angle. As such, the crystal angle was found to increase from about 24 degrees just below Tc to about 33 degrees below the pretransition. It was also observed that the P beta' superlattice did not form when annealed L beta' phase vesicles were heated from 5 degrees C to Tc; likewise, ripples did not form when the membrane was held under large tension during freezing from the L alpha phase. Each of these three procedures could be used to create a metastable planar structure which we have termed L*beta' since it is lamellar and plane-crystalline with acyl chains tilted to the bilayer plane. However, we show that this structure is not as condensed as the L beta' phase below 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental and theoretical studies of emodin interacting with a lipid bilayer of DMPC

Emodin is one of the most abundant anthraquinone derivatives found in nature. It is the active principle of some traditional herbal medicines with known biological activities. In this work, we combined experimental and theoretical studies to reveal information about location, orientation, interaction and perturbing effects of Emodin on lipid bilayers, where we have taken into account the neutral form of the Emodin (EMH) and its anionic/deprotonated form (EM^?). Using both UV/Visible spectrophotometric techniques and molecular dynamics (MD) simulations, we showed that both EMH and EM^? are located in a lipid membrane. Additionally, using MD simulations, we revealed that both forms of Emodin are very close to glycerol groups of the lipid molecules, with the EMH inserted more deeply into the bilayer and more disoriented relative to the normal of the membrane when compared with the EM^?, which is more exposed to interfacial water. Analysis of several structural properties of acyl chains of the lipids in a hydrated pure DMPC bilayer and in the presence of Emodin revealed that both EMH and EM^? affect the lipid bilayer, resulting in a remarkable disorder of the bilayer in the vicinity of the Emodin. However, the disorder caused by EMH is weaker than that caused by EM^?. Our results suggest that these disorders caused by Emodin might lead to distinct effects on lipid bilayers including its disruption which are reported in the literature.     

Skin preservation at 4 degrees C: a species comparison

There are numerous experimental studies in the literature regarding skin storage and preservation. These studies are difficult to interpret due to the variety of storage techniques utilized and the number of different animal species used as skin donors. This study utilized a single cold storage protocol to test the effect of species variation on skin graft viability. Donor skin was obtained from five animal species and human surgical panniculectomy specimens. The skin was stored in modified Roswell Park Memorial Institute (RPMI) 1640 tissue culture media at 4 degrees C. Stored skin was transplanted to surgically created defects on athymic (nude) mice after specific storage intervals. Ten days after transplantation, the grafts were examined by gross and microscopic techniques. The viability of mouse, rat, and dog skin was significantly different from human skin, while stored rabbit and pig skin were similar to human skin. These results demonstrate the difficulty of applying the data of skin storage studies from nonhuman species to clinical practice. The data indicate that rabbit and pig skin may be used in laboratory studies of skin preservation at 4 degrees C with a strong likelihood that the results may be of clinical relevance in predicting the behavior of human skin under similar storage conditions.     

Interfacial behavior of cholesterol, ergosterol, and lanosterol in mixtures with DPPC and DMPC

Binary mixtures of cholesterol, ergosterol, and lanosterol with phosphatidylcholines differing in the length of the saturated acyl chains, viz 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (DMPC), were analyzed using a Langmuir balance for recording force-area (pi-A) and surface potential-area (psi-A) isotherms. A progressive disappearance of the liquid expanded-liquid condensed transition was observed in mixed monolayers with DPPC after the increase in the content of all three sterols. For fluid DMPC matrix, no modulation of the monolayer phase behavior due to the sterols was evident with the exception of lanosterol, for which a pronounced discontinuity between mole fractions of X = 0.3 and X = 0.75 was discernible in the compression isotherms. Condensing and expanding effects in force-area (pi-A) isotherms due to varying X(sterols) and differences in the monolayer physical state were assessed from the values for the interfacial compression moduli. Surface potential measurements support the notion that cholesterol and ergosterol, but not lanosterol, reduce the penetration of water into the lipid monolayers. Examination of the excess free energy of mixing revealed an enhanced stability of binary monolayers containing cholesterol compared to those with ergosterol or lanosterol; the differences are emphasized in the range of surface pressure values found in natural membranes.     

Short-term storage of rabbit embryos at 4 degrees C

A simple method for storing preimplantation mammalian embryos was tested under conditions which could be easily maintained inside an ordinary refrigerator set at 4 degrees C. No significant loss of viability occurred when rabbit embryos were stored at 4 degrees C for 7 days and either cultured in vitro at 37 degrees C or transferred to recipient does. Significant losses occurred when embryos were stored for 10 days or longer before culture at 37 degrees C (P < .01). Stored embryos transferred to recipients had a significantly longer average gestation period than embryos transferred without cold storage (P < .05).     

Survival of bovine embryos stored at 4 degrees C

These experiments were designed to test the efficacy of storing bovine embryos at 4 degrees C. Of particular interest were the age of embryo at which maximum post-storage survival could be achieved and longevity at 4 degrees C. A greater proportion of day 8 blastocysts developed in vitro at 37 degrees C following refrigeration for 48 hr than did embryos collected 2, 4 or 6 days after estrus (P<0.01). Survival of blastocysts stored at 4 degrees C for 48 hr was similar to that of nonstored blastocysts. In a subsequent experiment, day 8 blastocysts were recovered nonsurgically and assigned to one of the following treatments: (a) immediate transfer; (b) culture at 37 degrees C; or (c) storage at 4 degrees C for 1, 2, 3 or 5 days. Post-storage viability was assessed by either development in culture at 37 degrees C or embryo survival following nonsurgical transfer to synchronized recipients. In vitro survival of nonstored embryos and embryos stored 1 day did not differ. Survival decreased after storage for 2 days (P<0.10) or longer (P<0.05). Similar results were observed for survival after transfer, but embryo viability decreased even more rapidly with increasing duration of storage. In vitro survival was approximately 50% for blastocysts stored for 3 and 5 days, but few pregnancies resulted from transfer of embryos stored for these periods. In another experiment survival after transfer of blastocysts stored at 4 degrees C for up to 2 days was similar to that of nonstored blastocysts.     

Interaction of aurein 1.2 and its analogue with DPPC lipid bilayer

Antibacterial peptides have potential as novel therapeutic agents for bacterial infections. Aurein 1.2 is one of the smallest antibacterial peptides extracted from an anuran. LLAA is a more active analogue of aurein 1.2. Antibacterial peptides usually accomplish their function by interacting with bacterial membrane selectively. In this study, we tried to find the reasons for the stronger antibacterial activity of LLAA compared with aurein 1.2. For this purpose, the interaction of aurein 1.2 and LLAA with dipalmitoylphosphatidylcholine (DPPC) was investigated by molecular dynamics (MD) simulation. In addition, the structure of peptides and their antibacterial activity were investigated by circular dichroism (CD) and dilution test method, respectively. MD results showed that LLAA is more flexible compared with aurein 1.2. Furthermore, LLAA loses its structure more than aurein 1.2 in the DPPC bilayer. A higher amount of water molecules penetrate into bilayer in the presence of LLAA relative to aurein 1.2. According to the antibacterial result that indicated LLAA is remarkably more active than aurein 1.2, it can be concluded that flexibility of the peptide is a determining factor in antibacterial activity. Probably, flexibility of the peptides facilitates formation of effective pores in the lipid bilayer.     

Thermotropic phase behaviour of the pseudobinary mixtures of DPPC/C12E5 and DMPC/C12E5 determined by differential scanning calorimetry and ultrasonic velocimetry

The present paper reports on the phase behaviour of the pseudobinary aqueous mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/pentaethylene glycol monododecyl ether (C12E5) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine monohydrate (DMPC)/C12E5. Both systems exhibit a variety of mesophases, such as lamellar gel, liquid crystalline and micellar phases. The phase diagrams show peritectic and eutectic behaviours. The existence of a compound complex is established. From the phase diagrams, the temperature dependence of the solubilisation parameters is obtained. The phase diagrams, especially with respect to the solubilisation process were qualitatively explained assuming that the packing of the constituents plays a dominating role. Finally, differential scanning calorimetry and ultrasonic velocimetry are compared concerning their potentials to determine characteristics of phase transitions in pseudobinary phospholipid/surfactant mixtures.     

Influence of DPH on the structure and dynamics of a DPPC bilayer

We have conducted extensive molecular dynamics (MD) simulations together with differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) experiments to quantify the influence of free 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescent probes on the structure and dynamics of a dipalmitoylphosphatidylcholine bilayer. Atomistic MD simulations show that in the membrane-water interface the influence of DPH is minor, whereas in the acyl-chain region DPH gives rise to major perturbations. In the latter case, DPH is found to influence a wide range of membrane properties, such as the packing and ordering of hydrocarbon tails and the lateral diffusion of lipid molecules. The effects are prominent but of local nature, i.e., the changes observed in the properties of lipid molecules are significant in the vicinity of DPH, but reduce rapidly as the distance from the probe increases. Long-range perturbations due to DPH are hence not expected. Detailed DSC and (2)H NMR measurements support this view. DSC shows only subtle perturbation to the cooperative behavior of the membrane system in the presence of DPH, and (2)H NMR shows that DPH gives rise to a slight increase in the lipid chain order, in agreement with MD simulations. Potential effects of other probes such as pyrene are briefly discussed.     

Molecular-dynamic simulation of DPPC bilayer in different phase state: Hydration and electric field distribution in the presence of Be<Superscript>2+</Superscript> cations

New molecular-dynamic topology of phosphatidylcholine bilayer (DPPC) in total atomic OPLS force field was developed and used to study the structural characteristics of liquid-crystalline and gel state of lipid bilayer in the absence and in the presence of Na⁺ and Be²⁺ cations adsorbed at the interface and different in their affinity. The parameters of bilayer geometry, the amount of surface water, and the electrostatic potential distribution were estimated quantitatively from the simulation in both phase states. The azimuthal angle of hydrocarbon chains was found nearly the same in the region of each monolayer in gel state. The amount of surface water decreases upon bilayer “freezing” mainly by loss of water molecules not participating in H-bonds between lipid headgroups. The cation adsorption was shown to have a small effect on these H-bonded water molecules, whereas Be²⁺ appeared to retain surface water in the bilayer upon its freezing. The electric potential distribution in the normal direction to the membrane-water interface had a similar shape in any bilayer phase state regardless of the presence of the adsorbed cations. Analysis of the microscopic nature of the electric potential revealed a mutual compensation of the contributions of the main structural components of the system (lipids, water, and ions). The boundary potential increased by 116 mV for pure DPPC, by 212 mV in the presence of Na⁺, and by 133 mV in the presence of Be²⁺ upon the phase transition of bilayer to the gel state. The boundary potential difference in the presence of Na⁺ and Be²⁺ and its change at the bilayer phase transition are in a good agreement with the experimental data published earlier [Ermakov Yu.A., 1993].     

In vitro survival of Echinococcus granulosus protoscolices in several media, at +4 degrees C and +37 degrees C

As a first step towards hydatid disease control, the in vitro survival of protoscolices of Echinococcus granulosus was investigated for various media and temperatures. Higher percentage survival was obtained than previously reported: at 4 degrees C, 100% survival was obtained for 20 days in medium 199 (GIBCO) and for 25 days in hydatid fluid from the host of origin. Maximal survival was 30% at 55 days in these conditions. Flame cell activity was the criterion of choice for viability. At 37 degrees C survival rates were lower and morphological changes in protoscolices were observed.     

Fertility of mouse spermatozoa retrieved from cadavers and maintained at 4 degrees C

After male animals die, the spermatozoa within the testis and epididymis eventually disintegrate. In this study, the motility, viability and fertility of mouse spermatozoa were examined after retrieval from the epididymis at various days after death. Cadavers were maintained in a refrigerator at 4 degrees C. About 30% of the spermatozoa collected 10 days after death were viable, but they had limited ability to fertilize oocytes in vitro. However, when the spermatozoa were injected into oocytes, the fertilization rate was over 80%. Normal live fetuses were even obtained using immotile spermatozoa retrieved 20 days after death. Therefore, when valuable male animals die unexpectedly and sperm cryopreservation is not possible immediately, temporal storage of cadavers (or epididymis and vas deferens) at 4 degrees C in a regular refrigerator followed by intracytoplasmic sperm injection may help to preserve the genome of individuals. This procedure could be particularly important in endangered species.     

Survival of ovine embryos stored at 4 degrees C for 24 hours

This study was conducted to ascertain if sheep embryos collected for transfer can be stored for short periods without freezing to allow for international transport. Of twelve Finnish Landrace ewes treated with equine follicle stimulating hormone (FSH), eleven ewes ovulated with a mean of 9.1 +/- 4.3 (SD) corpora lutea. Recovery rate from the nine ewes with normal corpora lutea was 68 +/- 27%, providing 61 morulae which were then cooled to 4 C and stored for 24 h while transporting them from Scotland to France. Romanov recipients received either 4 (n = 14) or 5 (n = 1) of these morulae. Fourteen of the recipients lambed, with a mean lambing rate of 2.1 +/- 0.8, representing 48.3% of embryos transferred. Cooling of embryos to 4 C and storing them in ovum culture medium for 24 h at 4 C may be a valuable technique for the handling and short-term storage of embryos.     

Effects of vitamin E on fowl semen storage at 4 degrees C

Vitamin E was assayed for either in chicken spermatozoa or seminal plasma. Effects of vitamin E on the motility and fertilizing ability of chicken semen stored for 24 hours at 4 degrees C were also studied. A mean of 0.25 mug vitamin E 10 (9) cells was found in spermatozoa and 0.074 mug in seminal plasma. When the medium for in vitro storage of semen was supplemented with vitamin E the motility of spermatozoa was not affected. However, vitamin E improved the fertilizing ability of semen stored for 24 hours at 4 degrees C, especially at the dose of 8 mug/ml of semen diluent.