Steven C. Hand

LEA Proteins During Water Stress: Not Just for Plants Anymore

Late embryogenesis abundant (LEA) proteins are extremely hydrophilic proteins that were first identified in land plants. Intracellular accumulation is tightly correlated with acquisition of desiccation tolerance, and data support their capacity to stabilize other proteins and membranes during drying, especially in the presence of sugars like trehalose. Exciting reports now show that LEA proteins are not restricted to plants; multiple forms are expressed in desiccation-tolerant animals from at least four phyla. We evaluate here the expression, subcellular localization, biochemical properties, and potential functions of LEA proteins in animal species during water stress. LEA proteins are intrinsically unstructured in aqueous solution, but surprisingly, many assume their native conformation during drying. They are targeted to multiple cellular locations, including mitochondria, and evidence supports that LEA proteins stabilize vitrified sugar glasses thought to be important in the dried state. More in vivo experimentation will be necessary to fully unravel the multiple functional properties of these macromolecules during water stress.

Cryoprotection of phosphofructokinase with organic solutes: Characterization of enhanced protection in the presence of divalent cations

Phosphofructokinase (PFK) purified from rabbit skeletal muscle is fully inactivated after being frozen in liquid nitrogen for 30 s and thawed. The addition of 500 mM trehalose, sucrose, or proline to the enzyme solution prior to freezing results in a recovery of over 70% of the original activity after thawing. Slightly less stabilization is imparted by maltose and 4-hydroxyproline whereas glucose, glycine, inositol, and glycerol at concentrations up to 500 mM are relatively ineffective at protecting PFK. With 50 mM trimethylamine-N-oxide, almost 50% of the prefreeze activity is recovered, and this same level of cryoprotection is noted at concentrations up to 500 mM. The addition of ionic zinc to enzyme-organic solute mixtures prior to freezing greatly enhances the cryoprotection imparted by all of the solutes tested. This effect is not simply due to the summation of the individual cryoprotective capacities of zinc and the organic solute because in many instances a great degree of cryoprotection is noted when each component is present at a concentration at which, by itself, it is totally ineffective. In the presence of a constant 50 mM organic solute concentration, freeze-thaw stabilization of PFK is increased as the concentration of zinc is increased. When the zinc concentration is held constant (0.6 mM) and organic solute concentration varied, the maximum cryoprotection, in most cases, is noted with less than 50 mM organic solute. At higher solute concentrations the degree of enhancement decreases such that with 500 mM organic solute the addition of zinc results in only a slight increase in protection. The addition of ionic copper, cadmium, nickel, and cobalt to trehalose-PFK solutions prior to freezing also increases the percentage of activity recovered after thawing. Magnesium, manganese, and calcium are ineffective in this respect.

Metabolic Dormancy in Aquatic Invertebrates

One major approach used by invertebrates for coping with extreme environmental conditions is to enter states of metabolic and developmental dormancy. The possession of a latent or resting stage is a common occurrence in the life cycle of organisms inhabiting inconsistent or ephemeral environments, and this phenomenon has been a source of long-standing speculation and fascination (cf. van Leeuwenhoek 1702). Considering the remarkable habitat diversity that exists among aquatic invertebrates, it is not surprising that the distribution of dormancy is broadly scattered phylogenetically. Resting stages have now been reported in virtually every major phylum of invertebrates, with the notable exception of Echinodermata. In some taxa, mature adult forms have the ability to enter states of rest, while in others, specialized resting forms are common only during earlier stages of life cycles.

Anaerobic Dormancy Quantified in Artemia Embryos: A Calorimetric Test of the Control Mechanism

Continuous measurement of heat dissipation from brine shrimp embryos during reversible transitions from aerobic development to anaerobic dormancy demonstrates a primary role for intracellular pH(pHi)in this metabolic switching. Artificially elevating the depressed pHi during anoxia by adding ammonia markedly reactivates metabolism, as judged by increases in heat dissipation, trehalose catabolism, and the ratio of adenosine triphosphate to adenosine diphosphate. Energy flow during anaerobic dormancy is suppressed to 2.4 percent of aerobic values, which is the lowest percentage thus far reported for euryoxic animals. Use of diguanosine tetraphosphate stores cannot account for this observed heat dissipation. Thus, mobilizing trace amounts of trehalose may explain the energy metabolism during quiescence.

Physical and chemical characteristics of ephemeral pond habitats in the Maracaibo basin and Llanos region of Venezuela

Physical and chemical variables of ephemeral rainwaterpond habitats in the Maracaibo basin and Llanos regionof Venezuela were investigated to assess environmentalfeatures important for future studies of thephysiological ecology and bioenergetics of annualkillifish. Dissolved oxygen, temperature, pH, Eh, andconductivity measurements were made in the field ateach collection site. Water and filter samples werecollected and analyzed for trace metals, cations,anions, and osmolality. Physical and chemicalcharacteristics of rainwater ponds are highly variableboth temporally and spatially. Large diurnalfluctuations occur in temperature and dissolvedoxygen. Dissolved oxygen content, temperature, pH andconductivity values exhibit a high degree of interpoolvariation. All pools sampled have a high amount ofsuspended solids consistent with high turbidity. Poolsediments appear to be anoxic as indicated bymeasurement of Eh. Llanos region pools are dominatedby calcium as the major cation, while Maracaibo basinpools are dominated by sodium as the major cation. Maracaibo pools can be further separated into twodistinct regions, inland savanna and coastal desert,based on ionic composition. Annual killifishinhabiting ephemeral ponds may be exposed to extremesin dissolved oxygen concentration, temperature and pHvalues during adult, juvenile, and embryonic stages.

Ontogeny of osmoregulation in the estuarine ghost shrimp Callianassa jamaicense var. louisianensis Schmitt (Decapoda, Thalassinidea)

During the abbreviated development of Callianassa jamaicense var. louisianensis Schmitt, both zoeal stages exhibit hyperosmotic regulation of hemolymph in media <800–900 mOsm and conform at higher salinities, a pattern similar to adults. Hyperosmotic regulatory ability is present at the time of hatching. (Na + K)-activated ATPase activity, commonly associated with salt-transporting tissues, increases from 7.8 ± 4.3 μmol Pi·h−1·g wet wt−1 (95 % CI, n = 7) at early stages of prehatch development to reach the highest levels at hatching (96.5 ± 10.6 μmol Pi·h−1·g wet wt−1, n = 21); thereafter, larval ATPase activity decreases and then stabilizes at 56.6 ± 12.4 μmol Pi·h−1·g wet wt−1 (n=21). AgNO3 stains potential sites for osmoregulation along the inside surface of the branchiostegite and adjacent body wall in the larvae by forming silver precipitates which are identified by energy dispersive X-ray analysis. TEM studies of these sites reveal a specialized epithelium with characteristics typical of salt-transporting tissue. Thus physiological, biochemical and morphological features present in these larvae reflect an early development of osmoregulatory ability.

Arrestment of carbohydrate metabolism during anaerobic dormancy and aerobic acidosis inArtemia embryos: determination of pH-sensitive control points

SummaryChanges in concentrations of trehalose, glycogen, glycerol, some glycolytic intermediates and adenylate nucleotides that occur during aerobic development have been compared to those seen during anaerobic dormancy and aerobic acidosis in gastrula-stage embryos ofArtemia. The latter two incubation conditions are known to foster large drops in intracellular pH (Busa et al. 1982; Busa and Crowe 1983).During aerobic development, trehalose levels decline while glycogen and glycerol are synthesized (Fig. 1). These transitions are blocked during both anaerobic dormancy and aerobic acidosis, but are resumed by return of embryos to aerobic incubation (Fig. 1). Thus, it is concluded that carbohydrate catabolism in hydrated embryos is directly modulated by intracellular pH. Changes in metabolite levels (Figs. 2–4) reveal that this process is controlled primarily at the trehalase and hexokinase reactions with a less-pronounced negative crossover point noted at the phosphofructokinase step. Each of these reactions is shown to be nonequilibrium by comparing the mass action ratio to the equilibrium constant (Table 1).When embryos are placed under anaerobic conditions, ATP levels drop dramatically while AMP increases in concentration (Fig. 5). These changes are reflected in a drop in adenylate energy charge from a control value of 0.73 to 0.42 (Fig. 6). Aerobic acidosis only leads to a slight decrease in energy charge, emphasizing that shifts in adenylate pools

Reversible Depression of Oxygen Consumption in Isolated Liver Mitochondria during Hibernation

The biochemical mechanisms by which hibernators cool as they enter torpor are not fully understood. In order to examine whether rates of substrate oxidation vary as a function of hibernation, liver mitochondria were isolated from telemetered ground squirrels (Spermophilus lateralis) in five phases of their annual hibernation cycle: summer active, and torpid, interbout aroused, entrance, and arousing hibernators. Rates of state 3 and state 4 respiration were measured in vitro at 25°C. Relative to mitochondria from summer‐active animals, rates of state 3 respiration were significantly depressed in mitochondria from torpid animals yet fully restored during interbout arousals. These findings indicate that a depression of ADP‐dependent respiration in liver mitochondria occurs during torpor and is reversed during the interbout arousals to euthermia. Because this inhibition was determined to be temporally independent of entrance and arousal, it is unlikely that active suppression of state 3 respiration causes entrance into torpor by facilitating metabolic depression. In contrast to the observed depression of state 3 respiration in torpid animals, state 4 respiration did not differ significantly among any of the five groups, suggesting that alterations in proton leak are not contributing appreciably to downregulation of respiration in hibernation.

Depression of Protein Synthesis during Diapause in Embryos of the Annual Killifish Austrofundulus limnaeus

Rates of protein synthesis are substantially depressed in diapause II embryos of Austrofundulus limnaeus. Inhibition of oxygen consumption and heat dissipation with cycloheximide indicates that 36% of the adenosine triphosphate (ATP) turnover in prediapausing embryos (8 d postfertilization [dpf]) is caused by protein synthesis; the contribution of protein synthesis to ATP turnover in diapause II embryos is negligible. In agreement with the metabolic data, incorporation of amino acids (radiolabeled via 14CO2) into perchloric acid–precipitable protein decreases by over 93% in diapause II embryos compared with embryos at 8 dpf. This result represents a 36% reduction in energy demand because of depression of protein synthesis during diapause. Adjusting for changes in the specific radioactivity of the free amino acid pool at the whole‐embryo level yields rates of protein synthesis that are artifactually high and not supportable by the observed rates of oxygen consumption and heat dissipation during diapause. This result indicates a regionalized distribution of labeled amino acids likely dictated by a pattern of anterior to posterior cell cycle arrest. AMP/ATP ratios are strongly correlated with the decrease in rates of protein synthesis, which suggests a role for adenosine monophosphate (AMP) in the control of anabolic processes. The major depression of protein synthesis during diapause II affords a considerable reduction in energy demand and extends the duration of dormancy attainable in these embryos.

Occurrence of Mitochondria-targeted Late Embryogenesis Abundant (LEA) Gene in Animals Increases Organelle Resistance to Water Stress

Anhydrobiotic animals survive virtually complete loss of cellular water. The mechanisms that explain this phenomenon are not fully understood but often include the accumulation of low molecular weight solutes such as trehalose and macromolecules like Late Embryogenesis Abundant (LEA) proteins. Here we report for the first time the occurrence of a mitochondria-targeted LEA gene (Afrlea3m) product in an animal species. The deduced molecular mass of the 307-amino acid polypeptide from the brine shrimp Artemia franciscana is 34 kDa. Bioinformatic analyses reveal features typical of a Group 3 LEA protein, and subcellular localization programs predict targeting of the mature peptide to the mitochondrial matrix, based on an N-terminal, amphipathic presequence. Real-time quantitative PCR shows that Afralea3m mRNA is expressed manyfold higher in desiccation-tolerant embryonic stages when compared with intolerant nauplius larvae. Mitochondrial localization of the protein was confirmed by transfection of human hepatoma cells (HepG2/C3A) with a nucleotide construct encoding the first 70 N-terminal amino acids of AfrLEA3m in-frame with the nucleotide sequence for green fluorescence protein. The chimeric protein was readily incorporated into mitochondria of these cells. Successful targeting of a protein to human mitochondria by use of an arthropod signaling sequence clearly reveals the highly conserved nature of such presequences, as well as of the import machinery. Finally, mitochondria isolated from A. franciscana embryos, which naturally contain AfrLEA3m and trehalose, exhibit resistance to water stress (freezing) as evidenced by an unchanged capacity for oxidative phosphorylation on succinate + rotenone, a resistance that is absent in mammalian mitochondria lacking AfrLEA3m.