The ability to adapt to altered availability of free water is a fundamental property of living cells. The principles underlying osmoadaptation are well conserved. The yeast Saccharomyces cerevisiae is an excellent model system with which to study the molecular biology and physiology of osmoadaptation. Upon a shift to high osmolarity, yeast cells rapidly stimulate a mitogen-activated protein (MAP) kinase cascade, the high-osmolarity glycerol (HOG) pathway, which orchestrates part of the transcriptional response. The dynamic operation of the HOG pathway has been well studied, and similar osmosensing pathways exist in other eukaryotes. Protein kinase A, which seems to mediate a response to diverse stress conditions, is also involved in the transcriptional response program. Expression changes after a shift to high osmolarity aim at adjusting metabolism and the production of cellular protectants. Accumulation of the osmolyte glycerol, which is also controlled by altering transmembrane glycerol transport, is of central importance. Upon a shift from high to low osmolarity, yeast cells stimulate a different MAP kinase cascade, the cell integrity pathway. The transcriptional program upon hypo-osmotic shock seems to aim at adjusting cell surface properties. Rapid export of glycerol is an important event in adaptation to low osmolarity. Osmoadaptation, adjustment of cell surface properties, and the control of cell morphogenesis, growth, and proliferation are highly coordinated processes. The Skn7p response regulator may be involved in coordinating these events. An integrated understanding of osmoadaptation requires not only knowledge of the function of many uncharacterized genes but also further insight into the time line of events, their interdependence, their dynamics, and their spatial organization as well as the importance of subtle effects.

Osmotic Stress Signaling and Osmoadaptation in Yeasts

Lyophilization and development of solid protein pharmaceuticals

Numerous organisms are capable of surviving more or less complete dehydration. A common feature in their biochemistry is that they accumulate large amounts of disaccharides, the most common of which are sucrose and trehalose. Over the past 20 years, we have provided evidence that these sugars stabilize membranes and proteins in the dry state, most likely by hydrogen bonding to polar residues in the dry macromolecular assemblages. This direct interaction results in maintenance of dry proteins and membranes in a physical state similar to that seen in the presence of excess water. An alternative viewpoint has been proposed, based on the fact that both sucrose and trehalose form glasses in the dry state. It has been suggested that glass formation (vitrification) is in itself sufficient to stabilize dry biomaterials. In this review we present evidence that, although vitrification is indeed required, it is not in itself sufficient. Instead, both direct interaction and vitrification are required. Special properties have often been claimed for trehalose in this regard. In fact, trehalose has been shown by many workers to be remarkably (and sometimes uniquely) effective in stabilizing dry or frozen biomolecules, cells, and tissues. Others have not observed any such special properties. We review evidence here showing that trehalose has a remarkably high glass-transition temperature (Tg). It is not anomalous in this regard because it lies at the end of a continuum of sugars with increasing Tg. However, it is unusual in that addition of small amounts of water does not depress Tg, as in other sugars. Instead, a dihydrate crystal of trehalose forms, thereby shielding the remaining glassy trehalose from effects of the added water. Thus under less than ideal conditions such as high humidity and temperature, trehalose does indeed have special properties, which may explain the stability and longevity of anhydrobiotes that contain it. Further, it makes this sugar useful in stabilization of biomolecules of use in human welfare.

The role of vitrification in anhydrobiosis.

Desiccation tolerance of prokaryotes.

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 sugars9 ability to lower the membrane phase transition temperature and to protect protein structure in the dry state.(ABSTRACT TRUNCATED AT 250 WORDS)

Samuel B. Leslie

Papers

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

Phase Transition Temperature and Chilling Sensitivity of Bovine Oocytes

Is Vitrification Sufficient to Preserve Liposomes during Freeze-Drying?

Trehalose lowers membrane phase transitions in dry yeast cells