Biomedical subjects

J H Crowe

Publications and source records attributed to J H Crowe.

At least 73 records · Page 4Linked to original sources

PubMed1991

Cord factor (alpha,alpha-trehalose 6,6'-dimycolate) inhibits fusion between phospholipid vesicles.

The persistence of numerous pathogenic bacteria important in disease states, such as tuberculosis, in humans and domestic animals has been ascribed to an inhibition of fusion between the phagosomal vesicles containing the bacteria and lysosomes in the host cells [Elsbach, P. & Weiss, J. (1988) Biochim. Biophys. Acta 974, 29-52; Thoen, C. O. (1988) J. Am. Vet. Med. Assoc. 193, 1045-1048]. In tuberculosis this effect has been indirectly attributed to the production of cord factor (alpha,alpha-trehalose 6,6'-dimycolate). We show here that cord factor is extraordinarily effective at inhibiting Ca2(+)-induced fusion between phospholipid vesicles and suggest a mechanism by which cord factor confers this effect. These findings are likely to be important in our understanding of the pathogenesis and treatment of many diseases of bacterial etiology.

Cord Factors↗

PubMed1991

Insights into the cryoprotective mechanism of dimethyl sulfoxide for phospholipid bilayers.

Dimethyl sulfoxide (Me2SO) is a widely used cryoprotectant for biological structures such as membranes. Despite hundreds of studies on the effects of this molecule, surprisingly little is known about its cryoprotective mechanism. This study investigates the ability of various Me2SO analogs to serve as cryoprotectants for liposomes. The data show that an increase in hydrophobicity progressively reduces the cryoprotective effect of sulfoxides. Additional experiments using phospholipid vesicles of varying composition demonstrate the Me2SO is markedly less effective on liposomes carrying a net negative charge. In fact, cryoprotection by Me2SO was virtually eliminated in vesicles composed of 30% phosphatidylserine (a negatively charged lipid). Based on these results, we suggest that the polar sulfoxide moiety of Me2SO interacts electrostatically with phospholipid membranes and that this interaction is critical for Me2SO's cryoprotective effect for membranes.

Binding Sites↗

PubMed1991

Effect of Sucrose on Phase Behavior of Membranes in Intact Pollen of Typha latifolia L., as Measured with Fourier Transform Infrared Spectroscopy.

In previous studies, we have shown that the temperature dependent vibrational frequency of the CH(2) stretch in hydrocarbons in intact pollen grains can be recorded with Fourier transform infrared spectroscopy and used to measure phase transition temperatures (T(m)) in these hydrocarbons. Circumstantial evidence was provided that the major contribution to the signal seen in these samples was from membrane phospholipids, and that sucrose in the dry pollen grains reduced T(m) of those phospholipids. In the current study, we clarify why a major constituent of the pollen grains, neutral lipids contained in discrete lipid droplets, does not contribute significantly to the signal. Further, we have isolated membranes from the pollen and show that T(m) in the isolated membranes rises from -6 degrees C in the hydrated membranes to 58 degrees C when the membranes are dried without the addition of sucrose. However, when the isolated membranes are dried in the presence of increasing amounts of sucrose, T(m) fell steadily, reaching a minimal value of 31 degrees C, a figure in good agreement with that seen in the intact pollen grains. The amount of sucrose required to depress T(m) maximally in these membranes is also apparently in agreement with that found in the intact pollen, suggesting that sucrose depresses T(m) in the pollen.

Journal Article↗

PubMed1991

Trehalose transport in yeast cells.

Saccharomyces cerevisiae possesses a specific transporter for trehalose. Transport is low until after glucose exhaustion in stationary phase, and addition of glucose in stationary phase results in loss of transport, an event that is reversible when the glucose is removed. The effect of glucose is not competitive inhibition: the increase in trehalose transport in stationary phase requires a lag time after glucose is depleted; and recovery of transport after glucose inhibition requires protein synthesis. Finally, we provide evidence that the trehalose transporter is distinct from that for maltose.

Binding, Competitive↗

PubMed1991

Trehalose-transporting membrane vesicles from yeasts.

We have isolated and characterized a membrane vesicle fraction from yeasts that is capable trehalose transport. The kinetics of the transport system were similar to those seen in the intact cells. The transport depends on a transmembrane pH gradient. If the gradient is collapsed, trehalose accumulated inside the vesicles is leaked into the medium. After aging for several days the ability of the vesicles to transport was lost. However, transport was partially restored by elevating internal pH in the vesicles.

Biological Transport↗

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↗

PubMed1989

An infrared spectroscopic study of the interactions of carbohydrates with dried proteins.

Fourier-transform infrared spectroscopy was used to characterize the interaction of stabilizing carbohydrates with dried proteins. Freeze-drying of trehalose, lactose, and myo-inositol with lysozyme resulted in substantial alterations of the infrared spectra of the dried carbohydrates. In the fingerprint region (900-1500 cm-1), there were large shifts in the frequencies of bands, a decrease in absorbance, and a loss of band splitting. These effects mimic those of water on hydrated trehalose. Bands assigned to hydroxyl stretching modes (around 3350 cm-1) were decreased in intensity and shifted to higher frequencies in the presence of the protein. In complementary experiments, it was found that dehydration-induced shifts in the positions of amide I and amide II bands for lysozyme could be partially and fully reversed, respectively, when the protein was freeze-dried in the presence of either trehalose or lactose. In addition, the carboxylate band, which was not detectable in the protein dried without the sugar, was apparent when these sugars were present. myo-Inositol was less effective at shifting the amide bands, and the carboxylate band was not detected in the presence of this carbohydrate. Also tested was the concentration dependency of the carbohydrates' influence on the position of the amide II band for dried lysozyme. The results showed that the ability of a given concentration of a carbohydrate to shift this band back toward the position noted with the hydrated protein coincided, at least in the extreme cases, with the capacity of that same level of carbohydrate to preserve the activity of rabbit skeletal muscle phosphofructokinase during freeze-drying.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrates↗

PubMed1989

Effects of free fatty acids and transition temperature on the stability of dry liposomes.

Previous studies have shown that liposomes composed of phospholipids with low phase-transition temperatures can be stabilized in the absence of water, provided that fusion is inhibited between the vesicles during drying, and that during rehydration the phospholipids do not pass through the gel to liquid crystalline phase transition. These conditions are met by adding certain disaccharides to the vesicles before drying, which inhibit fusion and depress the transition temperature in the dry lipids. The present study shows that preservation can also be achieved with vesicles made from dipalmitoylphosphatidylcholine (DPPC), but that the retention of trapped solute by such vesicles is much less than in vesicles composed of more fluid phospholipids. Addition of free fatty acids to the vesicles before drying destabilizes them; DPPC vesicles containing 15 mol% or more of palmitic acid leaked all their contents during drying, regardless of how much of the stabilizing sugar was added. Unlike the case for more liquid phospholipids, the leakage in DPPC vesicles is due solely to fusion and not to hydration-dependent phase transitions. Addition of free fatty acids results in increased fusion, leading to leakage.

Desiccation↗

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↗

PubMed1989

Lipid phase transitions measured in intact cells with Fourier transform infrared spectroscopy.

Lipid phase transitions in membranes are thought to be a major damaging event during cooling of cells prior to cryopreservation or during warming after freeze-thaw has been completed. Although there is abundant evidence that such transitions occur in isolated phospholipids, the evidence that they are found in membranes in intact cells is less clear, due largely to technical difficulties in detecting such transitions in the complex mixtures of lipids and proteins found in natural membranes. We show here that Fourier transform infrared spectroscopy provides a rapid, convenient method for detecting these transitions in intact cells. We have used intact pollen grains of cattail (Typha latifolia) as a primary experimental subject. Spectra taken of the intact pollen grains show most of the features commonly seen in natural membrane vesicles or pure phospholipids. Shifts in the vibrational frequency and width of the CH2 bands with temperature can be used to detect lipid phase transitions. Biochemical analysis, coupled with the spectroscopy, was used to assign transitions to nonpolar and polar lipids. Finally, although assignment of the melting lipid unambiguously in other cells has not yet been made, we show that the transitions can nevertheless be detected in other intact cells, including those of four plant species and sperm of three animals.

Freezing↗

PubMed1989

Membrane phase transitions are responsible for imbibitional damage in dry pollen.

We have found that the most probable cause of the leakage seen when dry cells or organisms such as seeds, pollen, or yeast cells are plunged into water is a gel to liquid crystalline phase transition in membrane phospholipids accompanying rehydration. By using Fourier transform infrared spectroscopy we have recorded infrared spectra of CH(2) stretching vibrations in dry and partially hydrated intact pollen grains of Typha latifolia. The vibrational frequency changes abruptly as phospholipids pass through the gel to liquid crystalline phase transition. Below the apparent transition, viable pollen shows low germination and high leakage when placed in water, but above the transition germination increases and leakage decreases. The apparent transition temperature falls with increasing water content, much as in pure phospholipids. By using this phenomenon, it was possible to construct a hydration-dependent phase diagram for the intact pollen. This phase diagram has immediate applications since it has high predictive value for the viability of the pollen when it is placed in water.

Journal Article↗

PubMed1988

Trehalose and dry dipalmitoylphosphatidylcholine revisited.

Dry mixtures of sonicated vesicles of DPPC and trehalose which contained a maximum of 0.2 mol water/mol lipid were examined by differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy. Samples of dry DPPC and trehalose prepared from aqueous solution had a minimum Tm of 24 degrees C for the gel to liquid-crystalline transition provided that the vesicles were dried with trehalose while the lipid was in liquid-crystalline phase. This low transition is compared to a transition of 105-112 degrees C for dry pure DPPC and of 42 degrees C for hydrated pure DPPC. The present work is an extension of earlier work from this laboratory using both other lipids and other methods of preparation.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1988

Mechanisms of interaction of amino acids with phospholipid bilayers during freezing.

In this study we compare the ability of various amino acids to protect small unilamellar vesicles against damage during freeze/thaw. Liposomes were composed of 75% palmitoyloleoyl phosphatidylcholine and 25% phosphatidylserine. Damage to liposomes frozen in liquid nitrogen and thawed at 20 degrees C was assessed by resonance energy transfer. Cryoprotection by numerous amino acids was compared in the presence and absence of 350 mM NaCl. The majority of amino acids with hydrocarbon side chains increased membrane damage during freeze/thaw regardless of the presence of salt. However, amino acids with hydrocarbon side chains of less than three carbons long, e.g. glycine, alanine, and 2-aminobutyric acid, were cryoprotective only in the presence of salt. We suggest that NaCl selectively increases the solubility of such amino acids, allowing them to act as cryoprotectants. In contrast, amino acids with side chains containing charged amine groups were cryoprotective regardless of the presence of salt. The degree of charge on the second amine group is shown to be important for cryoprotection by these molecules. We present evidence that suggests an interaction between the positively charged, second amine group of the amino acid, and the negatively charged phospholipid headgroup.

Amino Acids↗

PubMed1988

Interactions of sugars with membranes.

Water profoundly affects the stability of biological membranes, and its removal leads to destructive events including fusion and liquid crystalline to gel phase transitions. In heterogeneous mixtures such as those found in biological membranes the phase transitions can lead to increases in permeability and lateral phase separations that often are irreparable. Certain sugars are capable of preventing these deleterious events by inhibiting fusion during drying and by maintaining the lipid in a fluid state in the absence of water. As a result, the increased permeability and lateral phase separations that accompany dehydration are absent. The weight of the evidence suggests strongly that there is a direct interaction between the sugars and lipids in the dry state. Although the evidence is less clear about whether these sugars can interact directly with hydrated bilayers, there are strong suggestions in the literature that sugars free in solution or covalently linked to membrane constituents can also affect the physical properties and presumably the stability of bilayers. Finally, we have far less evidence concerning the mechanism by which they do so, but the same sugars are also capable of preserving the structure and function of both membrane-bound and soluble proteins in the absence of water. We believe these effects may be important in the survival of intact cells and organisms such as seeds in the absence of water. Furthermore, in view of the practical importance of preserving biological structures we suspect that the results described here will ultimately have important applications in biology and medicine.

Animals↗

PubMed1988

Factors affecting the stability of dry liposomes.

Previous studies have shown that liposomes can be preserved in the dry state in the presence of certain sugars, of which trehalose is particularly effective. There have been some discrepancies in results obtained by the various laboratories in which this phenomenon has been studied, both with respect to the efficacy of the sugars tested and the degree to which the dry vesicles can be stabilized. We show here that several factors that affect the stability of the dry liposomes may be responsible for the discrepancies between measurements by different laboratories. These factors include: (1) Size: small, sonicated vesicles are comparatively very unstable, and retain no more than 70% of trapped solute after drying, even in extremely high concentrations of sugars. Very large vesicles are similarly unstable. (2) Charge: a small amount of negatively charged lipid in the bilayer significantly increases stability. (3) Stabilizing sugar: the comparative efficacy of the sugar used varies with the size of the vesicles. (4) Dry-mass ratio. It is the dry-mass ratio between the stabilizing sugar and lipid that is important in the preservation during freeze-drying, not the concentration of either lipid or sugar in bulk solution.

Drug Stability↗

PubMed1988

Cryopreservation of sperm from the marine shrimp Sicyonia ingentis.

Sperm from a marine shrimp, Sicyonia ingentis, were frozen to -196 degrees C using a variety of cooling rates and cryoprotectants. A cooling rate of 1 degree C/min resulted in minimal cell breakage. Sperm samples were frozen in solutions of known membrane stabilizers--trehalose, sucrose, proline, and glycerol. These compounds were somewhat effective but a dramatic increase in sperm viability was seen when DMSO was present in the freezing medium. Sperm viability was assessed using the in vitro acrosome reaction technique of Griffin et al. (1987). The highest sperm survival (56%) was obtained with samples frozen at 1 degrees C/min in a 5% (v/v) DMSO solution. No decrease in viability was seen in sperm samples stored in liquid nitrogen (-196 degrees C) for 1 month.

Animals↗

PubMed1988

The mechanism of cryoprotection of proteins by solutes.

We have tested the capacity of 28 different compounds to protect lactate dehydrogenase from damage during freeze-thawing. These solutes come from very dissimilar chemical classes including sugars, polyols, amino acids, methylamines, and lyotropic salts. All the compounds tested, except NaCl, protected the enzyme, to varying degrees, from inactivation. The only characteristic that these compounds have in common, as a group, is that they have all been shown to be preferentially excluded from contact with the surface of proteins in aqueous solution. It has been demonstrated previously (via thermodynamic arguments) that this interaction of solutes with proteins leads to the stabilization of proteins in nonfrozen, aqueous systems. Conversely, those solutes, e.g., urea and guanidine HCl, that bind to proteins destabilize proteins in solution, and we have found that they also enhanced the inactivation of lactate dehydrogenase during freeze-thawing. Based on the results of our freeze-thawing experiments and a review of the theory of protein stabilization in nonfrozen, aqueous solution we propose that the cryoprotection afforded to isolated proteins by solutes can be accounted for by the fact that these solutes are preferentially excluded from contact with the protein's surface.

Animals↗

PubMed1988

Long-term preservation of dried phosphofructokinase by sugars and sugar/zinc mixtures.

We have demonstrated that sugars and suger/zinc mixtures can be used to preserve the activity of dried phosphofructokinase (PFK) during long-term storage over CaSO4. After 9 weeks in the presence of either 200 mM sucrose or 200 mM trehalose little loss of PFK activity was noted, with almost 60% of the original prefreeze-dry activity recovered when samples were rehydrated. Even reducing sugars protected the dried enzyme throughout the entire storage period. Of the sugars tested, 200 mM lactose provided the most stability to PFK; at the end of the dry storage, over 80% of the initial activity was recovered. With either 200 mM maltose or 400 mM glucose, about 40% of the initial activity was recovered at the end of the experiment. With all the sugars tested, the addition of 0.6 mM Zn2+ to sugar/PFK mixtures enhanced the stability of the enzyme, and no long-term adverse effects of the metal ion on enzyme activity were noted.

Animals↗