Phase Transition Of Membrane Lipids Research Articles

Cationic Proteins Rich in Lysine Residue Trigger Formation of Non-bilayer Lipid Phases in Model and Biological Membranes: Biophysical Methods of Study.

Cationic membrane-active toxins are the most abundant group of proteins in the venom of snakes and insects. Cationic proteins such as cobra venom cytotoxin and bee venom melittin are known for their pharmacological reactions including anticancer and antimicrobial effects which arise from the toxin-induced alteration in the dynamics and structure of plasma membranes and membranes of organelles. It has been established that these cationic toxins trigger the formation of non-bilayer lipid phase transitions in artificial and native mitochondrial membranes. Remarkably, the toxin-induced formation of non-bilayer lipid phase increases at certain conditions mitochondrial ATP synthase activity. This observation opens an intriguing avenue for using cationic toxins in the development of novel drugs for the treatment of cellular energy deficiency caused by aging and diseases. This observation also warrants a thorough investigation of the molecular mechanism(s) of lipid phase polymorphisms triggered by cationic proteins. This article presents a review on the application of powerful biophysical methods such as resonance spectroscopy (31P-, 1H-, 2H-nuclear magnetic resonance, and electron paramagnetic resonance), luminescence, and differential scanning microcalorimetry in studies of non-bilayer lipid phase transitions triggered by cationic proteins in artificial and biological membranes. A phenomenon of the triggered by cationic proteins the non-bilayer lipid phase transitions occurring within 10-2-10-11s is discussed in the context of potential pharmacological applications of cationic proteins. Next to the ATP dimer is an inverted micelle made of cardiolipin that serves as a vehicle for the transport of H+ ions from the intra-crista space to the matrix. It is proposed that such inverted micelles are triggered by the high density of H+ ions and the cationic proteins rich in lysine residue which compete with the conserved lysine residues of the ATP synthase rotor for binding to cardiolipin in the inner mitochondrial membrane and perturb the bilayer lipid packing of cristae. Phospholipids with a blue polar head represent cardiolipin and those with a red polar head represent other phospholipids found in the crista membrane.

Broad lipid phase transitions in mammalian cell membranes measured by Laurdan fluorescence spectroscopy

Employing fluorescence spectroscopy and the membrane-embedded dye Laurdan we experimentally show that linear changes of cell membrane order in the physiological temperature regime are part of broad order-disorder-phase transitions which extend over a much broader temperature range. Even though these extreme temperatures are usually not object of live science research due to failure of cellular functions, our findings help to understand and predict cell membrane properties under physiological conditions as they explain the underlying physics of a broad order-disorder phase transition. Therefore, we analyzed the membranes of various cell lines, red blood cell ghosts and lipid vesicles by spectral decomposition in a custom-made setup in a temperature range from −40 °C to +90 °C. While the generalized polarization as a measure for membrane order of artificial lipid membranes like phosphatidylcholine show sharp transitions as known from calorimetry measurements, living cells in a physiological temperature range do only show linear changes. However, extending the temperature range shows the existence of broad transitions and their sensitivity to cholesterol content, pH and anaesthetic. Moreover, adaptation to culture conditions like decreased temperature and morphological changes like detachment of adherent cells or dendrite growth are accompanied by changes in membrane order as well. The observed changes of the generalized polarization are equivalent to temperature changes dT in the range of +12 K < dT < -6 K.

Recent Progress to Explore the Mechanism of Direct Internalization of Cell-Penetrating Peptide (CPP) through BioMembrane

A specific series of peptides, called a cell-penetrating peptide (CPP), is known to be free to directly permeate through cell membranes into the cytosol (cytolysis); hence, this CPP would be a potent carrier for a drug delivery system (DDS). Previously, we proposed the mechanism of cytolysis as a temporal and local phase transfer of membrane lipid caused by positive membrane curvature generation. Moreover, we showed how to control the CPP cytolysis based on the mechanism. Here, we introduce key factors effectively modulate the CPC cytolysis so that they could be potent tools to control cytolysis. This short communication gives overview of our progressive investigation on the CPC cytolysis mechanism which inspired us to imagine many ways to control the cytolysis either to enhance or to suppress by maneuvering membrane curvatures corresponding to the temporal phase transitions of self-assembled membrane lipids under the liquid crystal states. We have shown the usefulness of physical stimuli such as osmotic pressure or particle size and chemical stimuli with their Hoffmeister effect to the outer medium by changing the hydration level of outer layers of membrane to control the CPP cytolysis by changing membrane curvature. Taking all the revealed vital factors and their effects as a tool, we will further explore how to control CPP cytolysis for developing a DDS system combined with appropriate cargo selection to be tagged with CPPs.

The Mechanisms of Action of Triindolylmethane Derivatives on Lipid Membranes.

The effects of new synthetic antibacterial agents – tris(1-pentyl-1H-indol-3-yl)methylium chloride (LCTA-1975) and (1-(4-(dimethylamino)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-indol-3-yl)bis(1-propyl- 1H-indol-3-yl)methylium chloride (LCTA-2701 – on model lipid membranes were studied. The ability of the tested agents to form ion-conductive transmembrane pores, influence the electrical stability of lipid bilayers and the phase transition of membrane lipids, and cause the deformation and fusion of lipid vesicles was investigated. It was established that both compounds exert a strong detergent effect on model membranes. The results of differential scanning microcalorimetry and measuring of the threshold transmembrane voltage that caused membrane breakdown before and after adsorption of LCTA-1975 and LCTA-2701 indicated that both agents cause disordering of membrane lipids. Synergism of the uncoupling action of antibiotics and the alkaloid capsaicin on model lipid membranes was shown. The threshold concentration of the antibiotic that caused an increase in the ion permeability of the lipid bilayer depended on the membrane lipid composition. It was lower by an order of magnitude in the case of negatively charged lipid bilayers than for the uncharged membranes. This can be explained by the positive charge of the tested agents. At the same time, LCTA-2701 was characterized by greater efficiency than LCTA-1975. In addition to its detergent action, LCTA-2701 can induce ion-permeable transmembrane pores: step-like current fluctuations corresponding to the opening and closing of individual ion channels were observed. The difference in the mechanisms of action might be related to the structural features of the antibiotic molecules: in the LCTA-1975 molecule, all three substituents at the nitrogen atoms of the indole rings are identical and represent n-alkyl (pentyl) groups, while LCTA-2701 contains a maleimide group, along with two alkyl substituents (n-propyl). The obtained results might be relevant to our understanding of the mechanism of action of new antibacterial agents, explaining the difference in the selectivity of action of the tested agents on the target microorganisms and their toxicity to human cells. Model lipid membranes should be used in further studies of the trends in the modification and improvement of the structures of new antibacterial agents.

The effect of lipid phase transitions in mitochondrial membranes on respiration and OxPhos system supercomplex formation

The effect of lipid phase transitions in mitochondrial membranes on respiration and OxPhos system supercomplex formation

Regulation of the Pore-Forming Activity of Cecropin A by Local Anesthetics

The influence of local anesthetics on the regulation of the channel-forming activity of the antimicrobial peptide cecropin A has been investigated. The mean current flowing through the single cecropin channels isc was determined, and steady-state transmembrane current induced by cecropin AI∞ was measured. It has been shown that the introduction of 1 mM of bupivacaine, benzocaine or 0.3 mM of tetracaine into the membrane bathing solution results in a decrease in isc and I∞. At the same time, the addition of 1 mM lidocaine or procaine to the membrane-bathing solutions does not lead to a significant change in isc and I∞. Comparison of the absolute values and the sign of the change in the boundary potential of negatively charged membranes and relative changes of isc and I∞ after addition of local anesthetics shows that neither parameter correlates with the membrane boundary potential. The results of studying the effect of tested local anesthetics on the phase transition of membrane lipids allow us to conclude that the observed changes of isc and I∞ are due to modulation of the elastic properties of the membrane.

Response of Сucumber (Cucumis sativus L.) Plants to Prolonged Permanent and Short-Term Daily Exposures to Chilling Temperature

We investigated the response of chilling-sensitive plants of cucumber (Cucumis sativus L.) to prolonged permanent (6 days) (PLT) and short-term (3 h) daily exposures to low non-freezing temperatures (9 and 12°С) (DROP) lying on different sides of the critical value (10°С) corresponding to a phase transition of membrane lipids in chilling-sensitive plants from a liquid-crystalline to a solid gel structure. Effects and aftereffects of DROP treatments at temperatures of 9 and 12°С (DROP9 and DROP12, respectively) were identical. They caused a reduction in linear dimensions of plants (as compared with control plants) and enhanced chilling tolerance of leaves but did not influence photosynthetic activity and water relations. However, when exposure to these temperatures was permanent (PLT9 and PLT12), their effects on plants were different. PLT9 almost entirely suppressed plant growth and development, inactivated photosynthetic apparatus (PSA), increased relative electrolite leakage (REL), and boosted lipid peroxidation (LPO). PLT12 also considerably reduced plant height and leaf area and suppressed (but did not inactivate) PSA; it did not boost POL or increased REL. It is important that, after the termination of chilling, PLT9 plants (unlike PLT12 plants) could not quickly resume growth and restore photosynthetic activity. Thus, considerable differences in plant responses to various patterns of chilling (long permanent or short-term daily) are probably due to the fact that, in the case of DROP treatments, relatively short-term (3 h) chilling of plants is followed in the diurnal cycle by a prolonged period (21 h) of optimal temperature when possible deviations and/or disturbances of PSA are restored and toxic substances that could accumulate in the course of chilling metabolized and/or neutralized. Pronounced differences in plant response to permanent exposure to temperatures of 9 and 12°С probably depend on the fact that these temperatures lie on different sides of a critical value (10°С) below which chilling-sensitive plants suffer grave physiological disturbances due to cooling. In addition, we showed that different responses of plants to PLT and DROP treatments are not determined by a usual dose-effect relationship but depend in many respects on the pattern of temperature influence (prolonged or short-term, single or recurring). As a result, the number of DROP spans experienced by plants in the experiments proved to be more important than their duration (at least, within a time interval from 2 to 12 h).

Plant Polyphenols Induced the Polymorphic Phase Transition of Membrane Lipids

Flavonoids are called “natural modifiers of biological reaction” due to their ability to change the response of the human body to allergens, viruses and oncogens. This is evidenced by their antioxidant, anti-inflammatory and antiparasitic and antibacterial properties. However, its use is limited by low relatively bioavailability. At the present time, the most perspective way to increase the bioavailability of polyphenols is used the complexes of modifiers with lipid vesicles. We had found that flavonoids (phloretin, butein, 4'-hydroxychalcone, naringenin, quercetin, myricetin, biochanin A, genistein, cardamonin, licochalcone A, liquiritigenin) are able to induce a polymorphic phase transition of DOPC. Large unilamellar liposomes were prepared by extrusion methods. The lipid:polyphenol ration was equal to 3:1, 1:1, and 1:3. Visualization of the polyphenol-induced non-bilayer lipid structures enriched with polyphenols were processed by laser scanning confocal microscope, Olympus FV3000. Phloretin, cardamonin, biochanin A, genistein, quercetin and myricetin caused the appearance of needle-shaped spherical non-bilayer structures, while the addition of butein and naringenin in the liposomal suspension leads to the appearance of rod-like structures. The morphology of non-bilayer structures induced by polyphenols does not depend on the ratio of lipid:polyphenol. The addition of 4'-hydroxychalcone, liquiritigenin and licochalcone A to the liposome suspension did not induced a polymorphic phase transition of lipids. Our results indicated a dependence of the polyphenol ability to induce the formation of non-bilayer structures on the number of OH groups in the molecule. It is proposed to apply the methods of electron microscopy and small-angle X-ray scattering to study the thin structure of the polyphenol-induced non-bilayer lipid structures. The study was supported in part by RFS (#17-74-10137).

СУЧАСНІ УЯВЛЕННЯ ПРО МЕТОДИ КРІОКОНСЕРВУВАННЯ ЕРИТРОЦИТІВ ТВАРИН

Nowadays, the method of blood transfusion is often used in veterinary practice. Hypothermic storage allows to save blood cells for a limited time, while morphofunctional parameters are getting worse. Cryopreservation allows to save and receive high-quality cells for the use in veterinary practice. Therefore, the development of reserves of donor blood is possible with long-term preservation in the frozen state. The use of cryopreservation makes it possible to avoid a number of problems: finding a donor at the right time for transfusion, the cost of maintaining the donor, etc. These days high therapeutic efficacy of using cryopreserved red blood cells was confirmed in intensive therapy and hematological diseases. The survival of biological objects under cryopreservation conditions is due to the ability of cells to withstand a complex of negative factors, including: the formation of crystals, an increase in the concentration of salts and osmotic pressure, dehydration of macromolecules and phase transitions of membrane lipids. Fundamental studies of physicochemical processes in cell suspensions under cooling and freezing conditions revealed important patterns that determine the basic principles of damage and protection of biological objects. The safety of cells under the influence of factors of low-temperature exposure is ensured by the use of special agents&nbsp; -&nbsp; cryoprotectants. Cryoprotective agents belong to different classes of organic compounds and are able to protect cells according to endo-and exocellular principle. However, the high efficiency of cryopreservation of red blood cells is achieved mainly by using endocellular compounds which must be removed after thawing from cell suspensions in order to maintain osmotic stability of cells under physiological conditions. Alternative to this may be cell-free cell cryopreservation methods developed on the basis of exocellular cryoprotectants. It should be noted that the cryoprotective properties of different compounds selectively manifest themselves depending on the type of cells. Biological preservation of erythrocytes for the use in veterinary practice is based on technologies for achieving biological stability and, accordingly, preserving a viable state after prolonged storage. This article reviews the mechanisms of cryodamage of erythrocytes, the cryoprotectants used in cryopreservation, as well as the existing low temperature storage methods.

Insights into composition/structure/function relationships of Doxil® gained from “high-sensitivity” differential scanning calorimetry

Thermotropic behavior of Doxil® and its generic, Lipodox®, was characterized using “high-sensitivity” differential scanning calorimetry (DSC). This is the first report that two distinct endotherms were observed in Doxil and Lipodox upon heating. The first (Tm at 51±2°C) is broad and of low enthalpy, representing the membrane lipid phase transition, which occurs despite having high (38mole%) cholesterol. The second (Tm at ∼70°C) is narrow, representing melting of the intraliposomal doxorubicin-sulfate nanocrystals. The thermograms of Doxil and Lipodox are practically identical. The membrane phase transition is similar to that of drug-free nanoliposomes of the same size and lipid composition as Doxil, suggesting lack of significant drug-membrane interaction. The melting endotherm of the intraliposomal nanocrystals is 2.0–2.5-fold narrower than that of the crystals formed in a solution of 250mM ammonium sulfate and >60mg/ml doxorubicin. This suggests that nanovolume of liposomes improves doxorubicin-sulfate crystallinity. Moreover, both phase transitions are reversible in cycled DSC scanning (15–90–15°C). This indicates an unexpected “non-leaky” phospholipid phase transition and excellent physical and chemical stabilities of Doxil after short exposure to high temperature. Reducing mole% of cholesterol results in a “leaky” membrane phase transition of higher enthalpy. Namely, high mole% cholesterol is essential for the resistance to drug leakage during phase transition. Pegylated liposomal doxorubicin in which HSPC was replaced by DPPC shows the same non-leaky phase transition but at a lower temperature, indicating this type of phase transition is not unique to Doxil. The presence of DSPE-PEG2k increases the cooperativity of the phase transition. High-sensitivity DSC helps illuminate composition/structure/function relationships of Doxil, and is useful for the equivalence/similarity studies.

16. Controlling cryoprotectant toxicity and chilling injury

Cryoprotectant toxicity and chilling injury are the central problems blocking the full deployment of vitrification, including its use for organ cryopreservation. Controlling these sources of injury is handicapped by the fact that little information is available about the nature of either cryoprotectant toxicity or chilling injury. To date, toxicity has been controlled by following 14 largely empirical rules of toxicity suppression in vitrification, but recently both theory and empirical experiment have begun to point the way to a coming ability to transcend cryoprotectant toxicity and with it, chilling injury as well. One interpretation of a recent gene expression profiling experiment on rat liver slices is that cryoprotectant toxicity in advanced vitrification solutions is related to protein denaturation. This is consistent with independent evidence that toxicity is correlated with stronger water hydrogen bonding by the permeating cryoprotectants in such solutions, which might destabilize the protein hydration layer and thus induce denaturation. Individual cryoprotectants apparently injure cells by specific mechanisms that don’t include denaturation, but when individual agents are diluted and combined to form vitrification solutions of high total concentration, injury may be primarily non-specific and mediated by denaturation linked to reduced water availability. Although worrisome at first, cells have evolved many ways of coping with protein denaturation, which may be augmented to achieve a therapeutic effect. In addition, chilling injury of vitrifiable liver slices was found to mimic cryoprotectant toxicity based on additional gene expression profiling, suggesting that chilling injury during vitrification may arise from the superimposition of cold denaturation on pre-existing protein destabilization by cryoprotectants. This implies that reducing cryoprotectant toxicity will also reduce chilling injury, but other interventions for chilling injury may also be helpful, including interventions that have been aimed at preventing membrane lipid phase transitions in other systems. Interestingly, those interventions include using antifreeze proteins to stabilize the cell membrane, which raises the question of whether other ice-active agents that might be more practically incorporated into vitrification solutions would have similar protective effects in addition to their intended role of interfering with ice formation. Another factor in controlling chilling injury has been tonicity adjustment. It has been reported that setting the nominal osmolality of non-penetrating species in a vitrification solution to 1.2–1.5 times isotonic minimizes chilling injury in rabbit renal cortical slices at all temperatures from −20 °C to −80 °C, below which chilling injury no longer increases. While helpful, this intervention places constraints on the amount of impermeant that can be used in vitrification solutions for the kidney. The mechanism of this effect is unknown, although it has been speculated that hypertonicity prevents injury otherwise caused by thermal contraction of cell membranes.

Lessons learned in andrology: looking at the spermatozoa in context.

Lessons learned in andrology: looking at the spermatozoa in context.

Membrane Lipid Phase Transition Behavior of Oocytes from Three Gorgonian Corals in Relation to Chilling Injury

The lipid phase transition (LPT) from the fluid liquid crystalline phase to the more rigid gel structure phase that occurs upon exposure to low temperatures can affect physical structure and function of cellular membranes. This study set out to investigate the membrane phase behavior of oocytes of three gorgonian corals; Junceela fragilis, J. juncea and Ellisella robusta,at different developmental stages after exposure to reduced temperatures. Oocytes were chilled to 5°C for 48, 96 or 144 h, and the LPT temperature (LPTT) was determined with Fourier Transform Infrared (FTIR) spectroscopy. The J. fragilis oocytes had a higher LPTT (∼23.0–23.7°C) than those of J. juncea and E. robusta oocytes (approximately 18.3–20.3°C). Upon chilling for 96 h at 5°C, the LPTTs of J. juncea and E. robusta oocytes in the early (18.0±1.0 and 18.3±0.6°C, respectively) and late (17.3±0.6 and 17.7±1.2°C, respectively) stages were significantly lower than those of J. fragilis oocytes (20.3±2.1 and 19.3±1.5°C for the early and late stages, respectively). The LPTTs of early stage gorgonian oocytes was significantly lower than those of late stage oocytes. These results suggest that the LPT of three gorgonian oocytes at different developmental stages may have been influenced by the phospholipid composition of their plasma membranes, which could have implications for their low temperature resistance.

A low membrane lipid phase transition temperature is associated with a high cryotolerance of Lactobacillus delbrueckii subspecies bulgaricus CFL1

The mechanisms of cellular damage that lactic acid bacteria incur during freeze-thaw processes have not been elucidated to date. Fourier transform infrared spectroscopy was used to investigate in situ the lipid phase transition behavior of the membrane of Lactobacillus delbrueckii ssp. bulgaricus CFL1 cells during the freeze-thaw process. Our objective was to relate the lipid membrane behavior to membrane integrity losses during freezing and to cell-freezing resistance. Cells were produced by using 2 different culture media: de Man, Rogosa, and Sharpe (MRS) broth (complex medium) or mild whey-based medium (minimal medium commonly used in the dairy industry), to obtain different membrane lipid compositions corresponding to different recovery rates of cell viability and functionality after freezing. The lipid membrane behavior studied by Fourier transform infrared spectroscopy was found to be different according to the cell lipid composition and cryotolerance. Freeze-resistant cells, exhibiting a higher content of unsaturated and cyclic fatty acids, presented a lower lipid phase transition temperature (Ts) during freezing (Ts=−8°C), occurring within the same temperature range as the ice nucleation, than freeze-sensitive cells (Ts=+22°C). A subzero value of lipid phase transition allowed the maintenance of the cell membrane in a relatively fluid state during freezing, thus facilitating water flux from the cell and the concomitant volume reduction following ice formation in the extracellular medium. In addition, the lipid phase transition of freeze-resistant cells occurred within a short temperature range, which could be ascribed to a reduced number of fatty acids, representing more than 80% of the total. This short lipid phase transition could be associated with a limited phenomenon of lateral phase separation and membrane permeabilization. This work highlights that membrane phase transitions occurring during freeze-thawing play a fundamental role in the cryotolerance of Lb. delbrueckii ssp. bulgaricus CFL1 cells.

A Microfluidic Study of Megakaryocytes Membrane Transport Properties to Water and Dimethyl Sulfoxide at Suprazero and Subzero Temperatures.

Megakaryocytes (MKs) are the precursor cells of platelets. Cryopreservation of MKs is critical for facilitating research investigations about the biology of this important cell and may help for scaling-up ex-vivo production of platelets from MKs for clinical transfusion. Determining membrane transport properties of MKs to water and cryoprotectant agents (CPAs) is essential for developing optimal conditions for cryopreserving MKs. To obtain these unknown parameters, membrane transport properties of the human UT-7/TPO megakaryocytic cell line were investigated using a microfluidic perfusion system. UT-7/TPO cells were immobilized in a microfluidic system on poly-D-lysine-coated glass substrate and perfused with various hyper-osmotic salt and CPA solutions at suprazero and subzero temperatures. The kinetics of cell volume changes under various extracellular conditions were monitored by a video camera and the information was processed and analyzed using the Kedem-Katchalsky model to determine the membrane transport properties. The osmotically inactive cell volume (V(b)=0.15), the permeability coefficient to water (Lp) at 37°C, 22°C, 12°C, 0°C, -5°C, -10°C, and -20°C, and dimethyl sulfoxide (DMSO; Ps) at 22, 12, 0, -10, -20, as well as associated activation energies of water and DMSO at different temperature regions were obtained. We found that MKs have relatively higher membrane permeability to water (Lp=2.62 μm/min/atm at 22°C) and DMSO (Ps=1.8×10(-3) cm/min at 22°C) than most other common mammalian cell types, such as lymphocytes (Lp=0.46 μm/min/atm at 25°C). This information could suggest a higher optimal cooling rate for MKs cryopreservation. The discontinuity effect was also found on activation energy at 0°C-12°C in the Arrhenius plots of membrane permeability by evaluating the slope of linear regression at each temperature region. This phenomenon may imply the occurrence of cell membrane lipid phase transition.

Protein–protein interactions in carotenoid triggered quenching of phycobilisome fluorescence in Synechocystis sp. PCC 6803

An inquiry into the effect of temperature on carotenoid triggered quenching of phycobilisome (PBS) fluorescence in a photosystem II-deficient mutant of Synechocystis sp. results in identification of two temperature-dependent processes: one is responsible for the quenching rate, and one determines the yield of PBS fluorescence. Non-Arrhenius behavior of the light-on quenching rate suggests that carotenoid-absorbed light triggers a process that bears a strong resemblance to soluble protein folding, showing temperature-dependent enthalpy of activated complex formation. The response of PBS fluorescence yield to hydration changing additives and to passing of the membrane lipid phase transition point indicates that the pool size of PBSs subject to quenching depends on the state of some membrane component.

Thermotropic Lipid Phase Transition and the Behavior of Hydrolytic Enzymes in the Kidney Cortex Brush Border Membrane

Functional interactions of lipids and proteins were examined in brush-border membranes isolated from the kidney cortex by studying the temperature dependence of the hydrolytic enzyme activities. A close relationship was observed for the membrane proteins and the thermotropic lipid phase transitions. Three lines of evidences were provided for such dependence: a) Arrhenius relationship of the membrane-bound enzyme activities, and the effect of temperature in native and partially delipidated membranes, b) differential scanning calorimetric study of the membrane lipid phase transitions in the native and delipidated membranes, multilamellar vesicles prepared from the membrane extracted lipids, and in vesicles from dimyristoyl phosphatidylcholine, and c) the excimer (dimer)-formation studies of the membrane extrinsic fluorescent probe, pyrene, and the resultant membrane microviscosity. The brush-border membranes were partially delipidated with BuOH and 2,2,2-trifluoroethanol. The functional interactions of the delipidated membranes, which were greatly lost on lipid removal, were largely restored by the addition of exogenous lipids in the reconstitution process, which indicate the critical dependence of the membrane integral proteins on the neighboring lipid molecules in the bulk lipid phase.

Proteomic Analysis of Hypothermically Preconditioned Endothelial Cells Reveals Comprehensive Protection from Hypothermic Injury

Homeothermic cell injury during ultraprofound hypothermia has been attributed to several mechanisms, including loss of ionic homeostasis, uncoupling of energy metabolism, lipid phase transitions in membranes, cytoskeletal depolymerization or oxidative stress. Hypothermic preconditioning (HPC; 25° C/72 h) optimally protects human coronary artery endothelial cells from injury at 0° C. We hypothesized that HPC protects cells from cold-induced injury by adaptive alterations in proteins that participate in the above processes. Confluent cells were cultured at 37° (control) or 25° C (HPC) for 72 h and whole cell lysates were created for proteomic analysis. 400 proteins were separated by 2-D gel electrophoresis and were matched between control and HPC samples. 43 proteins showed significant changes and were digested for peptide mass fingerprinting and identification. 21 identified proteins decreased significantly during HPC and 12 identified proteins increased. HPC-induced changes included a decrease in endothelial mitogens and enzymes involved in heme synthesis, oxidative stress and apoptosis. HPC increased the translation initiation factor eIF3 and stress proteins. Furthermore, changes in protein phosphatase-1 and ATP synthase levels and in intracellular chloride and potassium channels implicate a role for enhanced energy and volume regulation in HPC-treated cells. In conclusion, the overall pattern of HPC-induced changes reflects a comprehensive approach to protecting endothelial cells from injury at 0° C.

The effect of chilling on membrane lipid phase transition in human oocytes and zygotes

Chilling injury occurs when the cell membrane undergoes a transition from the liquid state to the gel state. Human oocytes and single-cell zygotes are of similar shape and size but the post-thawing survival rate of oocytes is poorer. We set out to investigate the possible difference in membrane lipid phase transition (LPT) temperature between the two cell types. The LPT temperature was measured with a Fourier Transform Infrared analyser, which detects the change in the vibration frequency of the CH2 bond stretches of the membrane lipid molecules during temperature change. The LPT temperatures of unfertilized human oocytes, in vitro-matured oocytes, and immature germinal vesicle (GV) stage oocytes were compared with that of abnormally fertilized human zygotes. The LPT temperatures of zygotes and of mature and immature GV oocytes differ significantly from each other (10.0 +/- 1.2, 16.9 +/- 0.9 and 24.4 +/- 1.6 degrees C respectively; P < 0.05). Zygotes show a higher resistance to chilling injury compared to oocytes at different developmental stages; this might explain the relatively poor survival rates of cryopreserved human oocytes and indicates the necessity to adjust the cryopreservation protocols in order to minimize cryoinjury.

Microbial inactivation by new technologies of food preservation

The increasing consumer demand for 'fresh-like' foods has led to much research effort in the last 20 years to develop new mild methods for food preservation. Nonthermal methods allow micro-organisms to be inactivated at sublethal temperatures thus better preserving the sensory, nutritional and functional properties of foods. The aim of this review is to provide an overview of the microbiological aspects of the most relevant nonthermal technologies for microbial inactivation currently under study, including irradiation, high hydrostatic pressure, pulsed electric field and ultrasound under pressure. Topics covered are the mechanisms of inactivation, sensitivity of different microbial groups and factors affecting it and kinetics of inactivation.