James S. Clegg

Desiccation Tolerance in Encysted Embryos of the Animal Extremophile, Artemia

Abstract Encysted embryos (cysts) of the primitive crustacean, Artemia franciscana, are among the most resistant of all animal life history stages to extremes of environmental stress. These embryos, extremophiles of the animal kingdom, are the main focus of this paper. Previous work has revealed the importance of biochemical and biophysical adaptations that provide a significant part of the basis of their resistance, and I consider some of these here. In the present paper the critical role played by the outer layer of the shell in desiccation tolerance will be one focus. Another involves studies on the response of dried cysts to high temperatures that, among other things, implicate one or more volatile factors released from the cysts that determines the extent of thermotolerance under a given heating regime. A hypothetical scheme is given to account for these peculiar results. Based on western immunoblotting analysis, and data from the literature, the scheme also implicates the heat-induced translocation of the stress protein p26 to nuclei as a potential cause of the reduction in hatching level.

FREE GLYCEROL IN DORMANT CYSTS OF THE BRINE SHRIMP ARTEMIA SALINA, AND ITS DISAPPEARANCE DURING DEVELOPMENT

1. Free glycerol was identified as a major carbohydrate component of the dormant cysts of Artemia salina.2. The amount of glycerol present in cysts aged for a year in the dry state was found to be about 5% of the cyst weight, and was shown to be restricted to the embryonic part of the cyst.3. Glycerol content decreased slightly during the formation of the nauplius and then rapidly decreased to a very low level after the nauplius emerged from the cyst. The decrease in glycerol content could not account for the synthesis of glycogen during formation of the nauplius.4. The glycerol, trehalose, and glycogen contents, and the viability of cysts aged up to 28 years were determined.

Anhydrobiosis: the water replacement hypothesis

Both the association of amphiphiles to form phospholipid bilayers and the folding of proteins that results in their tertiary structure are profoundly influenced by the low solubility of hydrocarbons in water (e.g. Tanford, 1978). These molecular arrangements, which are thought to be entropically driven, are lost when the water in which they are formed is removed. For instance, when a biological membrane is dehydrated, irreversible changes occur in its structural (Crowe and Crowe, 1982) and functional (Crowe, Crowe and Jackson, 1983) integrity. Similarly, many labile proteins lose their functional (reviewed in Carpenter, 1994) and probably structural (Prestrelski, Arakawa and Carpenter, 1993) integrity when they are desiccated. However, since the mid-1970s evidence has been accumulating that certain sugars may replace the water around polar residues in membrane phospholipids and proteins, maintaining their integrity in the absence of water. In this review we provide a current summary of what is known about the mechanism of these effects.

Protein Stability in Artemia Embryos During Prolonged Anoxia

Encysted embryos (cysts) of the brine shrimp, Artemia franciscana, are arguably the most stress-resistant of all animal life-history stages. One of their many adaptations is the ability to tolerate anoxia for periods of years, while fully hydrated and at physiological temperatures. Previous work indicated that the overall metabolism of anoxic embryos is brought to a reversible standstill, including the transduction of free energy and the turnover of macromolecules. But the issue of protein stability at the level of tertiary and quaternary structure was not examined. Here I provide evidence that the great majority of proteins do not irreversibly lose their native conformation during years of anoxia, despite the absence of detectable protein turnover. Although a modest degree of protein denaturation and aggregation occurs, that is quickly reversed by a brief post-anoxic aerobic incubation. I consider how such extraordinary stability is achieved and suggest that at least part of the answer involves massive amounts of a small heat shock protein (p26) that acts as a molecular chaperone, the function of which does not appear to require ribonucleoside di- or tri-phosphates.

Cellular Infrastructure and Metabolic Organization

Publisher Summary This chapter focuses on the suborganelle cytoplasm of animal cells. Cytoplasmic organization is much more extensive than the usual paradigm of cell structure and function. More or less spatially independent organelles are conventionally believed to be embedded in a viscous, highly concentrated, homogeneous solution commonly referred to as the cytosol which has also been considered to contain many of the enzymes of intermediary metabolism, the soluble enzymes. Cytosol was coined essentially as the 105,000 g supernatant obtained from centrifugation of homogenates. However, it soon was used to describe the volume among cytoplasmic structures in intact cells, and the majority of contemporary biologists often equate the in vitro and in vivo cytosols. Cytologists of the early 20th century recognized and usually emphasized the gel nature of cytoplasm. Much was made of sol-gel transformations in certain cellular (cytoplasmic) properties, but little connection was made during that period between the structure of cytoplasm and the metabolism that took place.

L-929 Cells Under Hyperosmotic Conditions Water, Na+, and K+

Changes in cell water content resulting from sorbitol addition to the environment of L-929 cells were evaluated gravimetrically using14C-labeled polyethylene glycol as a probe of extracellular space. Reductions in cell water were proportional to sorbitol supplements up to 0.6 molal, above which no further measurable decrease occurred. No volume regulation occurred for at least 1 h but the percentage of cell water lost was quickly regained when physiological conditions were restored. The amount of cell water lost because of a given hyperosmotic exposure was found to exceed the loss of cell volume. That discrepancy could be the result of an overestimation of extracellular space and/or an underestimation of cell volume reduction as a result of infolding of the cell surface. Na+ and K+ were also measured in cells of variable water content and volume: no significant change occurred in the amounts of these ions per cell, but large increases in total cell concentration resulted from hyperosmotic exposure. The sum of Na+ and K+ concentrations exceeds the total osmotic pressure of the medium indicating that an appreciable fraction of Na+ and K+ must be bound to fixed charges within the cells. The results are evaluated in the context of intracellular organization.