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At least 973 records · Page 54Linked to original sources
PubMed1993

Cold shock damage is due to lipid phase transitions in cell membranes: a demonstration using sperm as a model.

When cells are cooled to temperatures above the freezing point of water at rates greater than a few degrees per minute, they sustain irreversible injury. Reduction of this "cold shock" damage could increase the survival of animals and plants at low environmental temperatures and improve the cryopreservation of plant and animal cells. Leakage of solutes across membranes, associated with thermotropic phase transitions in membrane lipids, is thought to be responsible, but this hypothesis has not been tested directly. Using Fourier transform infrared spectroscopy (FTIR), we measured the lipid phase transitions in intact, living sperm, the animal cell in which cold shock has been studied most extensively. A shift in the CH2 absorbance peaks indicates the transition from liquid-crystalline to gel phase. The phase transition in sperm membranes occurred at a lower temperature for a marine shrimp than for the pig. In each case, potassium leakage, which is a hallmark of cold shock damage, increased abruptly near the end of the phase transition. Human sperm are quite resistant to cold shock, and an abrupt lipid phase transition was not detected. This phase behavior is typical of membranes containing a high proportion of cholesterol, and human sperm have an unusually high sterol content. High cholesterol levels are known to stabilize membranes during cooling. Overall, the lipid phase behavior was consistent with the temperature range over which cooling was damaging for pig and shrimp sperm, and the with the extent of damage produced in pig and human sperm. This is the first direct evidence that cold shock results from lipid phase transitions in cell membranes.

Animals↗
PubMed1998

Conformational and dynamic properties of a 14 residue antifreeze glycopeptide from Antarctic cod.

The 1H and 13C NMR spectra of a 14-residue antifreeze glycopeptide from Antarctic cod (Tetramatomnus borchgrevinki) containing two proline residues have been assigned. 13C NMR relaxation experiments indicate motional anisotropy of the peptide, with a tumbling time in water at 5 degrees C of 3-4 ns. The relaxation data and lack of long-range NOEs are consistent with a linear peptide undergoing significant segmental motion. However, extreme values of some coupling constants and strong sequential NOEs indicate regions of local order, which are most evident at the two ATPA subsequences. Similar spectroscopic properties were observed in the 16-residue analogue containing an Arg-Ala dipeptide added to the C-terminus. Molecular modeling also showed no evidence of long-range order, but the two ATPA subsequences were relatively well determined by the experimental data. These motifs were quite distinct from helical structures or beta turns commonly found in proteins, but rather resemble sections of an extended polyproline helix. Thus, the NMR data provide a description of the local order, which is of relevance to the mechanism of action of the antifreeze activity of the antifreeze glycopeptides as well as their ability to protect cells during hypothermic storage.

Amino Acid Sequence↗
PubMed1999

Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR by peroxynitrite.

Protein tyrosine phosphatases (PTPs) contain an essential thiol in the active site which may be susceptible to attack by nitric oxide-derived biological oxidants. We assessed the effects of peroxynitrite, nitric oxide, and S-nitrosoglutathione on the activity of three human tyrosine phosphatases in vitro. The receptor-like T-cell tyrosine phosphatase (CD45), the non-receptor-like tyrosine phosphatase PTP1B, and leukocyte-antigen-related (LAR) phosphatase were all irreversibly inactivated by peroxynitrite in less than 1 s with IC(50) values of </=0.9 microM. PTP inactivation was also seen with equivalent concentrations of peroxynitrite generated by SIN-1, indicating that bolus peroxynitrite and cogeneration of superoxide and nitric oxide were equipotent. Rate constants for peroxynitrite-mediated PTP inactivation were determined by competition with cysteine and were among the fastest rates yet seen for reaction of peroxynitrite with any biological molecules. The bimolecular reaction rates for CD45, LAR, and PTP1B were 2.0 x 10(8), 2.3 x 10(7), and 2.2 x 10(7) M(-1) s(-1), respectively. Inactivation by peroxynitrite was essentially irreversible as incubation with dithiothreitol (DTT) restored less than 10% of the original phosphatase activity. Prolonged treatment with 0.4 mM DETA NONOate, which generated a steady-state concentration of 2 microM nitric oxide, was only slightly inhibitory. S-Nitrosoglutathione (1.0 mM) inhibited PTPs by approximately 50% after 30 min and the inhibition was completely reversed by DTT. Nitrotyrosine immunoblots of peroxynitrite-treated PTP1B revealed that peroxynitrite completely inactivated PTP1B prior to the appearance of protein tyrosine nitration. Peroxynitrite anion is structurally similar to phosphate anion both in terms of molecular diameter and charge. Thus, the extreme vulnerability of these PTPs to peroxynitrite-mediated inactivation is consistent with attraction of peroxynitrite anion to the active site and subsequent oxidation of the essential thiolate. These findings suggest that any PTP possessing the CXXXXXR active-site sequence could potentially be inactivated by peroxynitrite in vivo resulting in a net increase in tyrosine phosphorylation and profound effects on phosphotyrosine-dependent signaling cascades.

Aniline Compounds↗
PubMed1993

A dependable method for the synthesis of [14C]trehalose.

A new method for the preparation of [14C]trehalose was developed, based on the ability of yeast cells to accumulate trehalose under stress. The method is simple and reliable. It utilizes a yeast strain in which the gene that encodes for phosphoglucoisomerase has been deleted. Thus, exogenously supplied glucose is not metabolized, but is instead converted to trehalose. The [14C]-trehalose obtained is pure, it is hydrolyzed by trehalase, and it is not susceptible to the action of alpha-glucosidase. The yield of this method is in the order of 35% of the [14C]glucose supplied.

Carbon Radioisotopes↗
PubMed1994

Is vitrification sufficient to preserve liposomes during freeze-drying?

It has been suggested that stabilization of liposomes and proteins during freeze-drying requires only that they be maintained in a vitrified (glassy) state. In the present paper we show that vitrification is indeed necessary. However, dextran, which exists as a glass at a higher temperature than does trehalose and thus might be expected to stabilize liposomes more effectively, preserves DPPC liposomes only when extremely large quantities of the dextran are added. Dextran does not stabilize egg PC liposomes and, in fact, inhibits the stabilizing effects of trehalose. Dextran also does not depress Tm in the dry phospholipids and shows no interaction with the polar headgroup, as assessed by infrared spectroscopy. Trehalose, by contrast, depresses Tm in dry egg PC by about 60 degrees C and depresses vibrational frequency of the phosphate in the polar headgroup to the frequency seen in the hydrated lipid, an effect we ascribe to hydrogen bonding between the sugar and the polar headgroup. We conclude that while vitrification may be required it is not in itself sufficient to preserve freeze-dried liposomes.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

Stabilization of dry membranes by mixtures of hydroxyethyl starch and glucose: the role of vitrification.

R. P. Goodrich and co-workers (1989, U.S. Patent 4,874,690; 1992, Proc. Natl. Acad. Sci. USA 89,967-971) have reported that red blood cells can be preserved in the dry state by addition of mixtures of hydroxyethyl starch (HES) and glucose. More recently, Spieles and co-workers (1996, Cryo-Lett. 17, 43-52) found that HES alone is insufficient to preserve the dry cells and concluded on this basis that the studies of Goodrich et al. were incorrect. In the present paper we revisit that suggestion, using liposomes as a model to study effects of HES and glucose on membrane stability. In previous studies we and others have established that liposomes can be stabilized in the dry state if they are dried in the presence of disaccharides. Monosaccharides have not been effective. Measurements of effects of glucose on phase transitions in the dry lipids and vibrational frequency of the phosphate headgroup suggest that glucose shows an interaction with dry egg phosphatidylcholine similar to that seen with disaccharides. Nevertheless, glucose does not inhibit fusion in liposomes during drying, and it does not prevent leakage. Hydroxyethyl starch, which has a very high glass transition (Tg), inhibits fusion in the dry liposomes, but it does not depress the liquid crystalline to gel phase transition temperature (Tm) in the dry phospholipids, does not cause a shift in the phosphate vibration indicative of hydrogen bonding of the sugar to the phosphate, and does not stop leakage of trapped carboxyfluorescein. However, if glucose is added to the HES-containing samples, the liposomes are stabilized, so long as the samples are maintained below the Tg of the mixture. If they are heated above that Tg they fuse and leak their contents. We conclude that both glass formation and depression of Tm in the dry lipids are required. The role of glass formation in stabilization during drying of liposomes appears to be inhibition of fusion.

Drug Stability↗
PubMed1999

Are lipid phase transitions responsible for chilling damage in human platelets?

In previous studies we have proposed that the well-known chilling-induced activation of human blood platelets can be ascribed at least in part to a thermotropic phase transition in membrane lipids. The evidence that this is the case is reviewed and amplified in this review, followed by an examination of the available physical data concerning phase transitions in lipid mixtures that mimic the mixture found in platelet membranes. Assuming complete mixing at all temperatures and equal contributions of the members of the mixture to the phase transition, the lipid mixture found in platelets should give values for Tm ranging from about 1 degrees C to about 16 degrees C, depending on the isomers present in the mixture. (The former value is not in agreement with the observed Tm, but the latter is in excellent agreement.) However, examination of the phase diagram for a binary pair of lipids found in platelet membranes shows that ideal mixing almost certainly does not occur; instead of a linear phase diagram, a convex one was obtained. This shape for the phase diagram, which would displace Tm to an unexpectedly elevated temperature, is in agreement with previously published phase diagrams for mixtures of this type. The prediction, based on thermodynamic properties of lipids found in the platelets, is that Tm will be displaced upward in more complex mixtures of the composition found in platelets, a prediction that requires experimental testing.

Blood Platelets↗
PubMed2001

The trehalose myth revisited: introduction to a symposium on stabilization of cells in the dry state.

This essay is an introduction to a series of papers arising from a symposium on stabilization of cells in the dry state. Nearly all of these investigations have utilized the sugar trehalose as a stabilizing molecule. Over the past two decades a myth has grown up about special properties of trehalose for stabilization of biomaterials. We review many of such uses here and show that under ideal conditions for drying and storage trehalose has few, if any, special properties. However, under suboptimal conditions trehalose has some distinct advantages and thus may remain the preferred excipient. We review the available mechanisms for introducing trehalose into the cytoplasm of living cells as an introduction to the papers that follow.

Animals↗
PubMed2001

Membrane reorganization during chilling: implications for long-term stabilization of platelets.

This essay is a review of the various biophysical and biochemical events that make up the factors responsible for platelet cold-induced activation. It describes the formation of large membrane domains composed of raft aggregates that occur during chilling and storage. It also presents strong evidence that platelet membranes undergo lateral phase separation during prolonged storage in the cold and suggests that raft aggregation and lateral phase separation are key events which must be obviated to stabilize platelets and store them either in the frozen or in the dry state.

Blood Platelets↗
PubMed1989

Phase transitions and permeability changes in dry membranes during rehydration.

Dry phospholipid bilayers are known to undergo transient changes in permeability during rehydration. In this review, we present evidence from which we suggest that this permeability change is due to a gel to liquid-crystalline phase transition accompanying rehydration. If the transition is avoided, as in lipids that remain in gel phase whether dry or rehydrated, the problem of leakage during rehydration is obviated, at least in part. Further, the evidence that the transition temperature for dry bilayers can be depressed by certain sugars is discussed. Finally, we show that these principles can be extended to intact cells. Using pollen grains as a model, we have measured the transition temperature for membrane phospholipids and show that the transition is correlated with physiological measurements including permeability changes and subsequent germination. From the Tm values taken from pollen grains at different water contents, we have constructed a phase diagram for the intact pollen that has high predictive value for physiological properties.

Cell Membrane Permeability↗
PubMed1983

Preservation of structural and functional activity in lyophilized sarcoplasmic reticulum.

Ca-transporting microsomes isolated from abdominal muscle of lobster were lyophilized in the presence and absence of trehalose. The dry membranes appeared with freeze fracture to collapse into cup-shaped structures embedded in a matrix of trehalose when lyophilized in the presence of trehalose. Upon rehydration, the dry membranes yielded vesicles that were morphologically indistinguishable from freshly prepared ones. These rehydrated vesicles also showed ATPase activity and Ca transport only slightly different from those activities in freshly prepared vesicles. The concentration of trehalose required to achieve this degree of stabilization is about 0.3 g trehalose/g membrane. When the membranes were dried at lower trehalose concentrations extensive fusion occurred between vesicles, along with lateral phase separations of membrane proteins and lipids. The rehydrated vesicles showed poor Ca uptake and coupling between ATPase activity and Ca uptake. The membranes may also be stabilized in the dry state by lyophilizing them in sucrose, but about three times as much sucrose is required as trehalose.

Animals↗
PubMed1984

Infrared spectroscopic studies on interactions of water and carbohydrates with a biological membrane.

Infrared spectroscopy was used to investigate the changes in bands assigned to phospholipids and proteins in dehydrated and rehydrated sarcoplasmic reticulum. The changes in CH2 and CH3 stretching bands, amide bands, and phosphate stretching bands are similar to shifts in frequency seen for those bands in phospholipid and protein preparations during thermotropic phase transitions and hydration. IR studies on dry trehalose-sarcoplasmic reticulum mixtures show similar results; with increasing trehalose concentration in the dry mixtures, amide and phosphate bands shift to frequencies characteristic of hydrated samples. Changes in bands assigned to OH deformations in the trehalose suggest that the interaction between the carbohydrate and membrane is by means of hydrogen bonding between these -OH groups and membrane components.

Animals↗
PubMed1985

Preservation of freeze-dried liposomes by trehalose.

One of the practical difficulties with the frequently proposed use of liposomes for delivery of water-soluble substances to cells in whole organisms is that liposomes are relatively unstable during storage. We have studied the ability of trehalose, a carbohydrate commonly found at high concentrations in organisms capable of surviving dehydration, to stabilize dry liposomes. With trehalose both inside and outside the bilayer, almost 100% of trapped solute was retained in rehydrated vesicles previously freeze-dried with 1.8 g trehalose/g dry phospholipid. Trehalose is very effective at inhibiting fusion between liposomes during drying, as assessed by freeze-fracture and resonance energy transfer between fluorescent probes incorporated into the bilayer. However, inhibition of fusion alone does not account for the preservation of the dry liposomes, since the concentration of trehalose required to prevent leakage is more than 10-fold that required to prevent fusion. We provide evidence that stabilization of the dry liposomes requires depression of transition temperature and consequent maintenance of the constituent lipids in the dry liposomes in a liquid crystalline phase.

Chromatography, Gel↗
PubMed1985

Interaction of carbohydrates with dry dipalmitoylphosphatidylcholine.

Interactions of six carbohydrates (trehalose, sucrose, glucose, raffinose, inositol, and glycerol) with dry dipalmitoylphosphatidylcholine (DPPC) were studied using differential scanning calorimetry (DSC) and infrared spectroscopy (ir) in order to elucidate the mechanism by which some of these carbohydrates preserve structural and functional integrity of dry membranes. Results with DSC showed that trehalose depressed the main transition temperature (Tmid) of dry DPPC below that of fully hydrated DPPC, and raised the enthalpy of that transition more than did addition of water. Results obtained with ir spectroscopy suggested a potential mechanism for this interaction. In the presence of most of the carbohydrates the ir spectrum for DPPC showed changes similar to those seen when water was added to dry DPPC, and the asymmetric P = O stretching band was diminished in intensity. The degree to which the carbohydrates tested affected the integrated intensity of this band and the Tmid was correlated with the ability of those carbohydrates to preserve dry membranes. Also, bands assigned to -OH deformations in the trehalose and other carbohydrates were depressed in the presence of DPPC. Based on these observations, it is suggested that the mechanism of interaction between the carbohydrate and lipid involves hydrogen bonding between -OH groups on the carbohydrate and the phosphate head group of the phospholipid. The only exceptions to this pattern are glycerol, which depresses Tmid of dry DPPC, and myo-inositol, which has no effect on Tmid or the ir spectrum of DPPC; neither carbohydrate can preserve dry membranes. It is suggested, based on ir spectroscopy and previous results with monolayer preparations, that glycerol interacts with phospholipids by a mechanism different from that shown by the other carbohydrates.

Calorimetry, Differential Scanning↗
PubMed1986

Effects of three stabilizing agents--proline, betaine, and trehalose--on membrane phospholipids.

We have studied the interaction between three compounds which accumulate in organisms under hydration stress--proline, betaine, and trehalose--and the membrane phospholipids dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), and dimyristoylphosphatidylethanolamine in bulk solution. Film balance studies reveal that these compounds increase the area/molecule of these lipids. Differential scanning calorimetry has been employed to investigate the effect these agents have on the gel-to-liquid crystalline phase transition of multilamellar and small unilamellar vesicles of DMPC, dipalmitoylphosphatidylcholine, and POPC:phosphatidylserine (90:10 mole ratio) in bulk solution. In the presence of 1 M proline, trehalose, or betaine, the midtransition temperature in small unilamellar vesicles is reduced (up to 7 degrees C in 1 M trehalose), and the transition broadened. In contrast, multilamellar vesicles of similar lipid composition show an increased transition temperature in the presence of the same concentration of these compounds. This result suggests that the inner lamellae in multilamellar vesicles may be dehydrated with only a few outer lamellae exposed to the protective compound. Finally, we have used stereomodels of phosphatidylcholine to investigate the mechanism of action of these agents. Hydrogen bonding of trehalose to the head group region results in an increase in the distance between head groups of 6.9 A. This amount of spreading compares well with data from the monolayer experiments which indicate that maximal spreading of DMPC monolayers by trehalose is 6.5 A. Molecular models of proline and betaine have also been constructed, and these models suggest potential interactions between these compounds and phosphatidylcholines. For the amphipath proline, this interaction may involve intercalation between phospholipid head groups.

Betaine↗
PubMed1986

Cryoprotection of phosphofructokinase with organic solutes: characterization of enhanced protection in the presence of divalent cations.

Phosphofructokinase (PFK) purified from rabbit skeletal muscle is fully inactivated after being frozen in liquid nitrogen for 30 s and thawed. The addition of 500 mM trehalose, sucrose, or proline to the enzyme solution prior to freezing results in a recovery of over 70% of the original activity after thawing. Slightly less stabilization is imparted by maltose and 4-hydroxyproline whereas glucose, glycine, inositol, and glycerol at concentrations up to 500 mM are relatively ineffective at protecting PFK. With 50 mM trimethylamine-N-oxide, almost 50% of the prefreeze activity is recovered, and this same level of cryoprotection is noted at concentrations up to 500 mM. The addition of ionic zinc to enzyme-organic solute mixtures prior to freezing greatly enhances the cryoprotection imparted by all of the solutes tested. This effect is not simply due to the summation of the individual cryoprotective capacities of zinc and the organic solute because in many instances a great degree of cryoprotection is noted when each component is present at a concentration at which, by itself, it is totally ineffective. In the presence of a constant 50 mM organic solute concentration, freeze-thaw stabilization of PFK is increased as the concentration of zinc is increased. When the zinc concentration is held constant (0.6 mM) and organic solute concentration varied, the maximum cryoprotection, in most cases, is noted with less than 50 mM organic solute. At higher solute concentrations the degree of enhancement decreases such that with 500 mM organic solute the addition of zinc results in only a slight increase in protection. The addition of ionic copper, cadmium, nickel, and cobalt to trehalose-PFK solutions prior to freezing also increases the percentage of activity recovered after thawing. Magnesium, manganese, and calcium are ineffective in this respect.

Amino Acids↗
PubMed1990

Effects of protein perturbants on phospholipid bilayers.

Series of alcohols, amides, ureas, and sulfoxides with increasingly longer hydrocarbon chains have been shown to lower progressively the thermal denaturation temperature of proteins. This effect is presumably due to a hydrophobic interaction between the solute and nonpolar domains of the protein. Theoretically, these interactions should occur between the solute and any macromolecular structure having a nonpolar region to which the solute has access. A recent review by Arakawa et al. has summarized evidence for such an interaction between organic solutes and proteins and suggested that these interactions are favored at higher temperatures. The present study investigates the effects of several classes of compounds on the stability of phospholipid vesicles. The results show that many compounds that are known to perturb protein function also destabilize phospholipid bilayers as reflected by solute-induced loss of vesicle contents.

Alcohols↗