Mechanisms associated with cellular desiccation tolerance of Artemia encysted embryos from locations around the world.

Using differential scanning calorimetry we demonstrated the presence of biological glasses and measured the glass transition temperatures (Tg) in dry encysted gastrula embryos (cysts) of the brine shrimp, Artemia, from eleven different locations, two of which provided cysts from parthenogenetic animals. Values for Tg were highest, by far, in Artemia franciscana cysts from the Mekong Delta, Vietnam (VN), these cysts having been produced from previous sequential inoculations into growth ponds of cysts from the San Francisco Bay, California, USA. Tg values for three groups of A. franciscana cysts were significantly higher than those of other cysts (except those of Artemia persimilis) studied here, as well as all other desiccation-tolerant animal systems studied to date. We also measured three stress proteins (hsc70, artemin and p26) in all these cysts as well as the total alcohol soluble carbohydrates (ASC), about 90% of which is the disaccharide trehalose, a known component of biological glasses. We interpret the results in terms of mechanisms involved with desiccation tolerance and, to some extent, with thermal conditions at the sites of cyst collection.

Artemin is an RNA-binding protein with high thermal stability and potential RNA chaperone activity.

Encysted embryos of the crustacean, Artemia franciscana, are among the most stress-resistant of all multicellular eukaryotes, due in part to massive amounts of p26, a small heat shock protein, that acts as a molecular chaperone. These embryos contain equally large amounts of another protein called artemin, of previously unknown function, that we report on here. Its thermal stability allows large-scale purification in about a day, using ammonium sulfate fractionation and incubation at 70 degrees C for 7 min, followed by gel filtration. The latter yields an artemin-RNA complex from which the pure protein, apo-artemin, was obtained by anion-exchange chromatography. We evaluated the possibility that artemin acts as a molecular chaperone for proteins, but obtained no evidence for that in vitro. The association of RNA with apo-artemin occurs at high temperatures and, although it is not yet clear whether artemin has a specific role as an RNA chaperone, it does bind non-polyadenylated RNAs which are then translated in vitro. Artemin-RNA is thermostable, some molecules resisting destruction after 30 min at 90 degrees C. The first order rate constant for denaturation and aggregation of artemin-RNA at 85 degrees C is 8.5 x 10(-3)min(-1), which compares well with other thermostable proteins of similar size ( approximately 500 kDa) such as the ferritins with which artemin has amino acid sequence similarity. The amount of artemin extracted from embryos that had been stored dry, under laboratory conditions, since 1951 is comparable to the amount in contemporary embryos, indicating its stability in situ, and supporting the in vitro heating studies.

Influence of trehalose on the molecular chaperone activity of p26, a small heat shock/alpha-crystallin protein.

Encysted embryos of the primitive crustacean Artemia franciscana are among the most resistant of all multicellular eukaryotes to environmental stress, in part due to massive amounts of a small heat shock/alpha-crystallin protein (p26) that acts as a molecular chaperone. These embryos also contain very large amounts of the disaccharide trehalose, well known for its ability to protect macromolecules and membranes against damage due to water removal and temperature extremes. Therefore, we looked for potential interactions between trehalose and p26 in the protection of a model substrate, citrate synthase (CS), against heat denaturation and aggregation and in the restoration of activity after heating in vitro. Both trehalose and p26 decreased the aggregation and irreversible inactivation of CS at 43 degrees C. At approximate physiological concentrations (0.4 M), trehalose did not interfere with the ability of p26 to assist in the reactivation of CS after heating, but higher concentrations (0.8 M) were inhibitory. We also showed that CS and p26 interact physically during heating and that trehalose interferes with complex formation and disrupts CS-p26 complexes that form at high temperatures. We suggest from these results that trehalose may act as a "release factor," freeing folding intermediates of CS that p26 can chaperone to the native state. Trehalose and p26 can act synergistically in vitro, during and after thermal stress, suggesting that these interactions also occur in vivo.

Nuclear p26, a small heat shock/alpha-crystallin protein, and its relationship to stress resistance in Artemia franciscana embryos.

The role of the small heat shock/alpha-crystallin protein, p26, in transcription in Artemia franciscana embryos was examined using isolated nuclei, containing either control or elevated levels of p26, in transcription run-on assays. Heat shock or anoxia in vivo and acid pH in vitro were used to transfer p26 into nuclei. The results suggest that parameters other than, or in addition to, p26 are responsible for the reduced transcription rates observed and that decreases in pHi are involved. In vivo experiments indicate that RNA synthesis and, to a lesser extent, protein synthesis are downregulated in intact embryos recovering from heat shock and that the precursor pool is not limiting. Confocal microscopy confirmed that p26 moves into nuclei in response to heat shock and anoxia in vivo, and to low pH in vitro, and indicated that the nuclear distribution of p26 is similar under all three conditions. We present evidence that unstressed (control) embryos containing p26 in all their nuclei will not hatch, even under permissive conditions, and propose that they are unable to terminate diapause. Potential nuclear targets of p26 chaperone activity are discussed.

Cryptobiosis--a peculiar state of biological organization.

David Keilin (Proc. Roy. Soc. Lond. B, 150, 1959, 149-191) coined the term 'cryptobiosis' (hidden life) and defined it as 'the state of an organism when it shows no visible signs of life and when its metabolic activity becomes hardly measurable, or comes reversibly to a standstill.' I consider selected aspects of the 300 year history of research on this unusual state of biological organization. Cryptobiosis is peculiar in the sense that organisms capable of achieving it exhibit characteristics that differ dramatically from those of living ones, yet they are not dead either, so one may propose that cryptobiosis is a unique state of biological organization. I focus chiefly on animal anhydrobiosis, achieved by the reversible loss of almost all the organism's water. The adaptive biochemical and biophysical mechanisms allowing this to take place involve the participation of large concentrations of polyhydroxy compounds, chiefly the disaccharides trehalose or sucrose. Stress (heat shock) proteins might also be involved, although the details are poorly understood and seem to be organism-specific. Whether the removal of molecular oxygen (anoxybiosis) results in the reversible cessation of metabolism in adapted organisms is considered, with the result being 'yes and no', depending on how one defines metabolism. Basic research on cryptobiosis has resulted in unpredicted applications that are of substantial benefit to the human condition and a few of these are described briefly.

Diguanosine nucleotide metabolism and the survival of artemia embryos during years of continuous anoxia.

Encysted embryos of the primitive crustacean, Artemia franciscana, are remarkably resistant to a variety of harsh environmental conditions, including continuous anoxia for periods of years at physiological temperatures and water contents. Previous study produced no evidence of an ongoing anoxic metabolism, suggesting that these embryos remained viable in spite of the lack of detectable free energy flow and biosynthesis. That seeming violation of a major axiom of cell biology and biochemistry prompted us to re-examine the nucleotide pool of encysted embryos during prolonged anoxia. We found that the nucleotide Gp(4)G, present initially in very large amounts, decreased slowly as anoxia continued over the 5.6-year period examined. Studies on other nucleotides and associated enzymes, including results from previous papers, provide a plausible metabolic pathway leading to the provision of ATP and GTP to meet the needs of endergonic processes in anoxic embryos. Exactly what those processes are is not obvious. One possibility involves the extensive anoxia-induced nuclear translocation of the stress protein, molecular chaperone p26, whose large molecular mass (approximately 500 kDa) most likely requires a supply of free energy to cross the nuclear envelope. Support for this possibility comes from our finding here that p26 is also a GTPase.

Long-term anoxia in encysted embryos of the crustacean, Artemia franciscana: viability, ultrastructure, and stress proteins.

Cells of encysted embryos of Artemia franciscana, the brine shrimp, are among the most resistant of all animal cells to extremes of environmental stress. We focus here on their ability to survive continuous anoxia for periods of years, during which their metabolic rate is undetectable. We asked whether their impressive tolerance was reflected in changes at the ultrastructural level. The ultrastructure of encysted embryos previously experiencing 38 days and 3.3 years of anoxia was compared with those not undergoing anoxia (controls). Rough endoplasmic reticulum was abundant in anoxic embryos, in spite of the absence of protein biosynthesis in their cells. Other cytoplasmic changes had occurred in the anoxic cells, but overall their structure was remarkably intact, in view of their 3 years of continuous anoxia. A major difference was the presence of abundant electron-dense granules in the nuclei of anoxic embryos; these were present but rare in nuclei of controls. Biochemical fractionation and Western immunoblotting confirmed previous observations that substantial amounts of the small heat shock/alpha-crystallin protein (p26) translocated into nuclei of anoxic embryos. We have no evidence that the dense granules contain this protein, but that remains a possibility. In contrast, and contrary to expectation, proteins of the hsp70 and 90 families did not undergo anoxia-induced nuclear translocation, an unusual result since such translocations have been widely observed in cells from a variety of organisms.

Molecular characterization of a small heat shock/alpha-crystallin protein in encysted Artemia embryos.

Molecular chaperones protect cells during stress by limiting the denaturation/aggregation of proteins and facilitating their renaturation. In this context, brine shrimp embryos can endure a wide variety of stressful conditions, including temperature extremes, prolonged anoxia, and desiccation, thus encountering shortages of both energy (ATP) and water. How the embryos survive these stresses is the subject of continuing study, a situation true for other organisms facing similar physiological challenges. To approach this question we cloned and sequenced a cDNA for p26, a molecular chaperone specific to oviparous Artemia embryos. p26 is the first representative of the small heat shock/alpha-crystallin family from crustaceans to be sequenced, and it possesses the conserved alpha-crystallin domain characteristic of these proteins. The secondary structure of this domain was predicted to consist predominantly of beta-pleated sheet, and it appeared to lack regions of alpha-helix. Unique properties of the nonconserved amino terminus, which showed weak similarity to nucleolins and fibrillarins, are enrichments in both glycine and arginine. The carboxyl-terminal tail is the longest yet reported for a small heat shock/alpha-crystallin protein, and it is hydrophilic, a common attribute of this region. Site-specific differences between amino acids from p26 and other small heat shock/alpha-crystallin proteins bring into question the functions proposed for some of these residues. Probing of Southern blots disclosed a multi-gene family for p26, whereas two size classes of p26 mRNA at 0.7 and 1.9 kilobase pairs were seen on Northern blots, the larger probably representing nonprocessed transcripts. Examination of immunofluorescently stained samples with the confocal microscope revealed that a limited portion of intracellular p26 is found in the nuclei of encysted embryos and that it resides within discrete compartments of this organelle. The results in this paper demonstrate clearly that p26 is a novel member of the small heat shock/alpha-crystallin family of proteins. These data, in concert with its restriction to embryos undergoing oviparous development, suggest that p26 functions as a molecular chaperone during exposure to stress, perhaps able to limit protein degradation and thus ensure a ready supply of functional proteins when growth is reinitiated.

Purification, structure and in vitro molecular-chaperone activity of Artemia p26, a small heat-shock/alpha-crystallin protein.

Encysted brine-shrimp gastrulae bring their metabolism to a reversible standstill during diapause and quiescence, demonstrating a remarkable resistance to unfavourable environmental conditions. For example, mortality of Artemia embryos under normal temperature and hydration is very low, even after two years of anoxia, and embryos commonly experience complete desiccation as part of their developmental program. Previous evidence from our laboratories indicated that p26, an abundant low-molecular-mass cyst-specific protein capable of translocation into the nucleus, may have a protective function in Artemia cysts. p26 was purified to apparent homogeneity and a continuous sequence of 141 of its amino acids was determined by peptide sequencing, revealing that it is a member of the small-heat-shock/alpha-crystallin family of proteins. As determined by molecular-sieve chromatography and sucrose-density-gradient centrifugation, native p26 is a multimer of about 27 monomers with a molecular mass of approximately 700 kDa. Inactivation of citrate synthase was less when the enzyme was heated in the presence rather than the absence of p26. Additionally, the renaturation of heat-inactivated citrate synthase was promoted by p26. These results indicated that p26 possesses molecular-chaperone activity, a property of other small heat-shock/alpha-crystallin proteins. Our findings demonstrate that p26 has the potential to protect the macromolecular components of Artemia embryos, either as they encyst or upon exposure to environmental extremes. Protection may depend upon the ability of p26 to function as a molecular chaperone.

Nuclear-cytoplasmic translocations of protein p26 during aerobic-anoxic transitions in embryos of Artemia franciscana.

Embryos of the crustacean Artemia franciscana survive continuous anoxia for periods of years, during which their metabolism comes to a reversible stand-still. A question of some interest concerns the maintenance of cellular integrity in the absence of biosynthesis and an ongoing energy metabolism. The present paper continues previous work on an abundant protein (p26) that undergoes extensive intracellular translocation during aerobic-anoxic transitions, exhibits several characteristics of stress proteins, and might be involved in metabolic regulation during aerobic-anoxic transitions. Since it has been established that intracellular pH (pHi) plays a major role in aerobic-anoxic transitions in this system we examined the pH-dependence of nuclear-cytoplasmic translocations of p26. In unincubated and aerobic-incubated embryos (pHi > or = 7.9) p26 was located in the "soluble" fraction, whereas in anoxic embryos (pH about 6.3) roughly 50% was translocated into the nucleus as shown by immunocolloidal gold electron microscopy. These nuclear translocations also took place in vitro, simply by manipulating buffer pH in a physiologically appropriate fashion. Immunostaining of Western blots prepared after two-dimensional electrophoresis revealed several isoforms of native p26. The isoelectric point of the major isoform was 7.10 +/- 0.05, a value close to the pH at which p26 translocation into the nucleus was first initiated in vitro. 31P-NMR measurements indicated that pHi was maintained at acidic levels (about 6.3) during prolonged anoxia. We also found that pHi of hydrated (0 degree C) but otherwise unincubated embryos was alkaline, allowing for rapid resumption of metabolism under permissive conditions. The significance of these pH-dependent translocations of p26 is discussed.

Extensive intracellular translocations of a major protein accompany anoxia in embryos of Artemia franciscana.

Cells of encysted gastrula embryos of the crustacean Artemia franciscana exhibit extraordinary stability during prolonged anoxia. We find that they contain an abundant protein (referred to as "26-kDa protein") that undergoes translocation to the nucleus during anoxia. The reverse translocation rapidly occurs when anoxic embryos are returned to aerobic conditions. A similar translocation appears to take place in embryos exposed to 42 degrees C aerobic heat shock and prolonged exposure to low temperature (0-2 degrees C), and in diapause embryos. Gel filtration and Western immunoblotting indicate that the 26-kDa protein is translocated to other cellular compartments and may also be associated with a wide variety of "soluble" proteins during anoxia. This protein makes up roughly 15% of the total nonyolk embryo protein and is, by far, most abundant in the encysted embryo stage of the life cycle. The hypothesis is advanced that the 26-kDa protein may play the role of a metabolic regulator and/or a protective molecular chaperone during prolonged anoxia and other forms of stress.

What determines the basal metabolic rate of vertebrate cells in vivo?

Do vertebrate cells dictate basal metabolic rate or does the organism have some influence over this decision? In this paper we advance the idea that the rate of delivery of essential nutrients to cells could be a key regulatory mechanism, a concept which originates for Coulson (Comp. Biochem, Physiol., 84A, 1986, 217-229) that we have extended to the delivery of substrates to enzymes at the intracellular level.

Aerobic heat shock activates trehalose synthesis in embryos of Artemia franciscana.

Encysted embryos (cysts) of the brine shrimp, Artemia franciscana, contain large amounts of trehalose which they use as a major substrate for energy metabolism and biosynthesis for development under aerobic conditions at 25 degrees C. When cysts are placed at 42 degrees C (heat shock) these pathways stop, and the cysts re-synthesize the trehalose that was utilized during the previous incubation at 25 degrees C. Glycogen and glycerol, produced from trehalose at 25 degrees C, appear to be substrates for trehalose synthesis during heat shock. Anoxia prevents trehalose synthesis in cysts undergoing heat shock. These results are consistent with the view that trehalose may play a protective role in cells exposed to heat shock, and other environmental insults, in addition to being a storage form of energy and organic carbon for development.

Evidence for cooperativity of protein dissolution in Brij 58 permeabilized L929 cells.

Mouse L929 cells were exposed to the nonionic detergent Brij 58. As has been shown in some other cell types, protein leaked from Brij 58 exposed cells only after a lag phase. In the current study we have extended the observations of the kinetics of protein efflux using cultured L cells subjected to treatment with buffers containing Brij 58. The results show that while the cells become permeable essentially at first exposure to the detergent, proteins do not escape immediately. This lag in efflux is at least partly dependent on the concentration of detergent such that a greater lag is seen in cells exposed to the lowest concentrations of Brij. Data are presented that are most readily interpreted as protein leakage having occurred fairly rapidly from individual cells and that show that the time course of protein efflux results, to a large extent, from different sensitivities of individual cells to the detergent. The permeabilized suspension cells consist of only two types, whereas the conversion of cells from one type to the other occurs through the loss of protein to the permeabilization medium. Only two bands are seen in continuous density gradients and there is a conversion of the more dense type to the less dense with longer exposure to detergent. Moreover, the less dense cells contained about half of the protein per cell as the bottom banding cells, and the proteins of the more dense cells appear to be the sum of those released into the permeabilization medium plus those found in the less dense cells.

Glucose metabolism and the channeling of glycolytic intermediates in permeabilized L-929 cells.

L-929 cells (mouse fibroblasts) permeabilized with dextran sulfate (DSP cells) carry out vigorous and linear rates of glycolysis when supplied with a suitable incubation medium. Glycolysis in DSP cells is pH dependent, being strongly inhibited at pH 6.5. Compared to their nonpermeabilized counterparts, DSP cells exhibit faster glycolytic rates, but tend to convert a smaller proportion of the glucose utilized to lactate. [14C]Glucose is converted to lactate by DSP cells without dilution from endogenous substrates. When exogenous 12C-labeled glycolytic intermediates (12C-I) are added to glycolyzing DSP cells the [14C]lactate produced from [14C]glucose is diluted to varying extents, depending on the intermediate. However, the extent of that dilution (reduced specific activity) is not that expected from the complete mixing of exogenous 12C-I with their corresponding 14C-labeled intermediates coming from [14C]-glucose. DSP cells also respire and convert glucose to CO2. The amount of 14CO2 produced from [14C]glucose is also reduced by addition of most 12C-I, an interesting exception being pyruvate, which had no measurable effect on 14CO2 production and caused only a modest stimulation of respiration in glycolyzing DSP cells. These results suggest that channeling, or some other form of coupling, takes place between the glycolytic production of pyruvate and its further oxidation. These observations confirm previously published data and add further support to the proposition that channeling of glycolytic intermediates occurs in DSP cells but is of the "leaky" type. Although abundant evidence in the literature indicates that various glycolytic enzymes associate with F-actin, as well as other elements of the cytomatrix, we observed no effect of cytochalasin D on lactate production even at very high concentrations of this compound. Our results are compared with those from other laboratories and discussed in the context of metabolic organization.