Biomedical subjects

L M Crowe

Publications and source records attributed to L M Crowe.

At least 37 records · Page 2Linked to original sources

PubMed1995

Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying.

The microorganisms Escherichia coli DH5 alpha and Bacillus thuringiensis HD-1 show an increased tolerance to freeze-drying when dried in the presence of the disaccharides trehalose and sucrose. When the bacteria were dried with 100 mM trehalose, 70% of the E. coli and 57% of the B. thuringiensis organisms survived, compared with 56 and 44%, respectively, when they were dried with sucrose. Only 8% of the E. coli and 14% of the B. thuringiensis organisms survived drying without the sugars. Fourier transform infrared spectroscopy was used to investigate the role of membrane phase transitions in the survival of the organisms during drying and rehydration. Both E. coli and B. thuringiensis showed an increase of 30 to 40 degrees C in the temperature of their phospholipid phase transition when dried without the sugars, while phase transition temperatures of those dried with the sugars remained near those of the hydrated cells. A Fourier transform infrared spectroscopy microscope made it possible to investigate the effects of drying on the protein structure in the intact cells. The amide II peak shifts from 1,543 cm-1 in the hydrated cells to about 1,533 cm-1 in the cells dried without sugar. There is no shift in the amide II peak when the cells are dried with trehalose or sucrose. We attribute the increased survival to the sugars' ability to lower the membrane phase transition temperature and to protect protein structure in the dry state.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus thuringiensis↗

PubMed1994

Interaction of cord factor (alpha, alpha'-trehalose-6,6'-dimycolate) with phospholipids.

We previously reported that cord factor (alpha,alpha'-trehalose-6,6'-dimycolate) isolated from Nocardia asteroides strain GUH-2 strongly inhibits fusion between unilamellar vesicles containing acidic phospholipid. We chose to study the effects of this molecule on liposome fusion since the presence of N. asteroides GUH-2 in the phagosomes of mouse macrophages had been shown to prevent phagosomal acidification and inhibit phagosome-lysosome fusion. A virtually non-virulent strain, N. asteroides 10905, does not prevent acidification or phagosome-lysosome fusion and, further, contains only trace amounts of cord factor. In the present paper, we have investigated the effects of cord factor on phospholipid bilayers that could be responsible for the inhibition of fusion. We show that cord factor increases molecular area, measured by isothermal compression of a monolayer film, in a mixed monolayer more than would be expected based in its individual contribution to molecular area. Cord factor, as well as other glycolipids investigated, increased the overall hydration of bilayers of dipalmitoylphosphatidylcholine by 50%, as estimated from the unfrozen water fraction measured by differential scanning calorimetry. The effect of calcium on this increased molecular area and headgroup hydration was measured by fluorescence anisotropy and FTIR spectroscopy of phosphatidylserine liposomes. Both techniques showed that cord factor, incorporated at 10 mol%, increased acyl chain disorder over controls in the presence of Ca2+. However, FTIR showed that cord factor did not prevent headgroup dehydration by the Ca2+. The other glycolipids tested did not prevent either the Ca(2+)-induced chain crystallization or headgroup dehydration of phosphatidylserine bilayers. These data point to a possible role of the bulky mycolic acids of cord factor in preventing Ca(2+)-induced fusion of liposomes containing acidic phospholipids.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1994

Trehalose lowers membrane phase transitions in dry yeast cells.

Recent work has clearly demonstrated a direct correlation between the amount of trehalose present in the yeast Saccharomyces cerevisiae and its ability to tolerate dehydration, but has failed to elucidate the specific role played by trehalose. By using Fourier transform infrared spectroscopy we measured the transition temperature of phospholipids in both intact S. cerevisiae and isolated plasma membranes dried in the presence and absence of trehalose. Our results show that trehalose lowers the temperature of the dry gel to liquid crystal phase transition in yeast from around 60 degrees C to about 40 degrees C, thus allowing yeast rehydrated above 40 degrees C to avoid the damaging effects of passing through a phase transition. These results explain both the need for trehalose and the observation that yeast must be rehydrated with warm water if they are to remain viable. Only when trehalose is present is the dry transition within a physiologically tolerable range and only when the cells are rehydrated above 40 degrees C will they avoid passing through a phase transition.

Cell Membrane↗

PubMed1994

Trehalose inhibits ethanol effects on intact yeast cells and liposomes.

The effect of ethanol on stability of intact yeast cells has been investigated. Several strains with differences in trehalose metabolism were examined for their ability to survive in the presence of 10% (v/v) ethanol. A positive correlation was observed between cell viability and trehalose concentration. When leakage of electrolytes from the cells was recorded by observing changes in conductivity of the medium, we found that ethanol increases leakage, but the presence of trehalose reverses that effect. Similar studies were done with liposomes of similar composition to those seen in intact cells in log and stationary phases. In the presence of ethanol, carboxyfluorescein trapped in the liposomes leaked to the medium. When trehalose was added inside, outside or on both sides of the membrane, the ethanol-induced leakage was strongly inhibited. More leakage was observed in liposomes in gel phase state than in liquid-crystalline phase, suggesting that the thermotropic behavior of the lipids in the plasma membrane, together with trehalose, plays a role in enhancing ethanol tolerance.

1,2-Dipalmitoylphosphatidylcholine↗

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↗

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↗

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↗

PubMed1992

Temperature-dependent perturbation of phospholipid bilayers by dimethylsulfoxide.

Dimethylsulfoxide (DMSO) is known to protect isolated enzymes during freezing while destabilizing proteins at high temperatures. This apparent paradox is the subject of a review by Arakawa et al. ((1990) Cryobiology 27, 401-415), who present evidence for a temperature-dependent, hydrophobic interaction between DMSO and non-polar moieties of proteins. The present study investigates the interaction of DMSO with phospholipid bilayers. Phospholipid vesicles containing carboxyfluorescein were exposed to several concentrations of DMSO at various temperatures. Leakage rates increased with DMSO concentration and temperature. This effect was not reduced in the presence of solutes that have been shown to neutralize DMSO toxicity in tissues. The increased leakage rates correlate well with the increased partitioning of DMSO from water to octanol at higher temperatures. Additionally, reductions in the CH2 vibrations of the bilayer are also shown to depend on DMSO concentration and temperature. A similar reduction in CH2 vibrations was observed in solutions of octanol and DMSO, suggesting that this effect is not mediated through an interaction with water. Furthermore, investigation of sulfoxide vibrations indicate that DMSO is not hydrogen bonded to the alcohol moiety of octanol, and therefore the interaction between DMSO and octanol is most likely due to a hydrophobic association. These results are consistent with a destabilization of phospholipid membranes at higher temperatures due to a hydrophobic association between DMSO and the bilayer.

Dimethyl Sulfoxide↗

PubMed1992

Anhydrobiosis: a strategy for survival.

Many organisms from a wide variety of taxa have the ability to survive extreme dehydration, a phenomenon called "anhydrobiosis." Concomitantly with resistance to the adverse effects of drying, these organisms are also resistant to the effects of freezing to very low temperatures, elevated temperature for brief periods, and the effects of ionizing radiation. One result of their resistance to environmental extremes is a greatly prolonged life span. The anhydrobiotes that have been investigated share a common metabolic adaptation, the production of certain disaccharides as a large proportion of their dry weight. Using these disaccharides, we have investigated the sources of damage attendant upon drying and the mechanisms by which anhydrobiotes and model systems of isolated membranes and proteins avoid damage. This report summarizes aspects of this work.

Adaptation, Biological↗

PubMed1992

Anhydrobiosis.

We believe we have established the major principles governing the stabilization of living cells in the unique condition known as anhydrobiosis. These findings have permitted us to design ways to stabilize membrane vesicles, liposomes, and proteins, and perhaps eventually even intact cells that do not normally survive dehydration. In a complex phenomenon as ancient as anhydrobiosis, one would expect a myriad of adaptations to be required for survival of drying. But the arguments presented here suggest that a single perturbation--synthesis of a disaccharide such as trehalose or sucrose--is sufficient to achieve survival. We hasten to add, however, that it is now certain that additional adaptations are required; for instance, cells containing highly unsaturated lipids may survive drying for a short time, but they are so susceptible to degradation that they survive for a short time only. Thus the interpretation placed on the finding that trehalose can stabilize dry membranes must be regarded from this perspective as well. Nevertheless, we believe that the underlying physical principles governing stability of dry biological materials are universal.

Adaptation, Physiological↗

PubMed1992

Stabilization of dry liposomes by carbohydrates.

Many organisms have adopted biochemical strategies that enable them to survive dehydration, a condition known as "anhydrobiosis". In addition to resistance to the deleterious effects of drying, anhydrobiotes are also resistant to a number of other adverse environmental conditions. Studies on the biochemistry of anhydrobiotes show a strong correlation between their ability to survive drying and the production of specific sugars, of which the most widely studied is trehalose. Studies of model systems show that direct interactions of trehalose with dry biological membranes, liposomes, and isolated proteins avoid the damaging events caused by drying. Cellular components are maintained in a physical state when dry that mimics the hydrated state. Although the presence of only two disaccharides, trehalose and sucrose, has been correlated with survival of drying under natural conditions, other molecules that achieve the same results may be effective.

Carbohydrates↗

PubMed1991

Alterations in membrane surfaces induced by attachment of carbohydrates.

We have examined the behavior of the dry phospholipid dipalmitoylphosphatidylcholine (DPPC) in the presence of several carbohydrate derivatives. These carbohydrate derivatives possess a hydrophobic portion which is incorporated directly into the DPPC membrane and a hydrophilic portion which places the carbohydrate structure at the membrane interface with the surrounding matrix. In the presence of these derivatives, the physical properties of the membrane are altered. These alterations are evident in changes observed in the phosphate and carbonyl vibrational modes of the phospholipid portion of the membrane. In addition, the phase transition behavior of the lipid is significantly altered as evidenced by a reduction in the gel to liquid-crystalline phase transition temperature. These results are consistent with those previously reported for free carbohydrates interacting with membranes in which a water replacement hypothesis has been used to explain the behavior. The attachment of carbohydrates to the membrane enhances these effects by localizing the agent responsible for these alterations at the membrane interface.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1991

Solution effects on the thermotropic phase transition of unilamellar liposomes.

We have investigated the effect of two monosaccharides, glucose and fructose, and two disaccharides, sucrose and trehalose, on the thermotropic phase transition of unilamellar extruded vesicles of DPPC. All the sugars investigated raise the main transition temperature (Tm) of some fraction of the lipid, but there are differences between the effect of glucose and the other three sugars. At low concentrations of glucose, Tm is lowered. At high concentrations of glucose there are two transitions, one with a low Tm and one with a high Tm. The data suggest that at low concentrations, all of the glucose present may bind to the bilayer and increase headgroup spacing by physical intercalation or increased hydration. The appearance of a Tm above that of pure hydrated DPPC suggests the possibility of the dehydration of some other population of phospholipid molecules. The other three sugars increase Tm, but at high concentrations of trehalose, sucrose, and fructose a second peak occurs at a low Tm. The other sugars appear to dehydrate the bilayer at low concentrations, but may show some binding or increased hydration of some portion of the lipid at very high concentrations. The sugar effects on unilamellar vesicles are strikingly different from the effects of these sugars on multilamellar vesicles.

1,2-Dipalmitoylphosphatidylcholine↗

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↗