Profiles of nuclear and mitochondrial encoded mRNAs in developing and quiescent embryos of Artemia franciscana.

Embryos of the brine shrimp Artemia franciscana are able to withstand long bouts of environmental anoxia by entering a quiescent state during which metabolism is greatly depressed. Recent evidence supports a global arrest of protein synthesis during quiescence. In this study we measured the amounts of mRNA for a mitochondrial-encoded subunit of cytochrome c oxidase (COX I) and for nuclear-encoded actin during aerobic development, anaerobiosis, and aerobic acidosis (artificial quiescence imposed by intracellular acidification under aerobic conditions). The levels of both COX I and actin transcripts increased significantly during aerobic development. COX I mRNA levels were tightly correlated with previous measures of COX catalytic activity, which suggests that COX synthesis could be regulated by message concentration during aerobic development. The ontogenetic increase for these mRNAs was blocked by anoxia and aerobic acidosis. Importantly, the levels of COX I and actin mRNA did not decline appreciably during the 6 h bouts of quiescence, even though protein synthesis is acutely arrested by these same treatments. Thus, the constancy of mRNA levels during quiescence indicate that reduced protein synthesis is not caused by message limitation, but rather, is likely controlled at the translational level. One advantage of this regulatory mechanism is the conservation of mRNA molecules during quiescence, which would potentially favor a quick resumption of translation as soon as oxygen is returned to the embryos. Finally, because anoxia and aerobic acidosis are both characterized by acidic intracellular pH, the reduction in pH may serve, directly or indirectly, as one signal regulating levels of mRNA in this embryo during quiescence.

Acute blockage of the ubiquitin-mediated proteolytic pathway during invertebrate quiescence.

Many organisms withstand adverse environmental conditions by entering a reversible state of quiescence that may last for months or years. In this report we provide evidence that the reduction in adenylate energy status and the associated intracellular acidosis occurring during anoxia-induced quiescence combine to inhibit, directly or indirectly, the initial step in the ubiquitin-mediated proteolytic pathway in embryos of the brine shrimp Artemia franciscana. The levels of ubiquitin-conjugated proteins drop to 37% of control (aerobic) values during the first hour of anoxia and reach 7% in 24 h. ATP falls to 5% of control values under anoxia, and AMP rises reciprocally. This energy limitation is accompanied by a simultaneous depression of intracellular pH (pHi). By comparison, when embryos are subjected to artificial acidosis under aerobic conditions (pHi drops sharply, but ATP does not change for hours), ubiquitin-conjugated proteins decline to 58% after 1 h. Thus, while the proximate mechanism for the suppression of ubiquitination has not been proven, alterations in the adenylate pool and the decrease in pHi both appear to contribute to the suppression of ubiquitination. Western blot analysis indicates that the decline in ubiquitin-conjugated protein is rapidly reversed on return of embryos to control conditions. We conclude that this arrest of ubiquitination likely serves to suppress ubiquitin-mediated degradation of protein, thereby preserving macromolecular integrity and potentially explaining the remarkable extension of protein half-life observed under anoxia in these embryos.

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.

Extension of enzyme half-life during quiescence in Artemia embryos.

Encysted gastrulae of Artemia franciscana are known to enter a reversible state of quiescence in which biosynthetic and catabolic pathways are markedly suppressed. Given that these embryos can survive months of anoxia, we investigated their ability to extend the half-life of cytochrome-c oxidase (COX), a key metabolic enzyme, during anoxia. We calculate that the half-life of COX is extended to 101 days under anoxia, an estimated 77-fold increase compared with aerobic values. During conditions of aerobic acidosis, the half-life of COX was extended sevenfold to a value of 9.7 days. We propose that the extended lifetimes of COX in both cases may be due to suppressed mitochondrial proteolysis under depressed pH. The shorter enzyme half-life observed under the latter condition may be due to the availability of ATP for degradative processes during aerobic acidosis. We also suggest that the presence of oxygen in aerobic acidosis may lead to increased rates of protein damage due to autooxidation.

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.

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.

Effects of protein perturbants on phospholipid bilayers.

Series of alcohols, amides, ureas, and sulfoxides with increasingly longer hydrocarbon chains have been shown to lower progressively the thermal denaturation temperature of proteins. This effect is presumably due to a hydrophobic interaction between the solute and nonpolar domains of the protein. Theoretically, these interactions should occur between the solute and any macromolecular structure having a nonpolar region to which the solute has access. A recent review by Arakawa et al. has summarized evidence for such an interaction between organic solutes and proteins and suggested that these interactions are favored at higher temperatures. The present study investigates the effects of several classes of compounds on the stability of phospholipid vesicles. The results show that many compounds that are known to perturb protein function also destabilize phospholipid bilayers as reflected by solute-induced loss of vesicle contents.

Lipid phase transitions measured in intact cells with Fourier transform infrared spectroscopy.

Lipid phase transitions in membranes are thought to be a major damaging event during cooling of cells prior to cryopreservation or during warming after freeze-thaw has been completed. Although there is abundant evidence that such transitions occur in isolated phospholipids, the evidence that they are found in membranes in intact cells is less clear, due largely to technical difficulties in detecting such transitions in the complex mixtures of lipids and proteins found in natural membranes. We show here that Fourier transform infrared spectroscopy provides a rapid, convenient method for detecting these transitions in intact cells. We have used intact pollen grains of cattail (Typha latifolia) as a primary experimental subject. Spectra taken of the intact pollen grains show most of the features commonly seen in natural membrane vesicles or pure phospholipids. Shifts in the vibrational frequency and width of the CH2 bands with temperature can be used to detect lipid phase transitions. Biochemical analysis, coupled with the spectroscopy, was used to assign transitions to nonpolar and polar lipids. Finally, although assignment of the melting lipid unambiguously in other cells has not yet been made, we show that the transitions can nevertheless be detected in other intact cells, including those of four plant species and sperm of three animals.

Interactions of sugars with membranes.

Water profoundly affects the stability of biological membranes, and its removal leads to destructive events including fusion and liquid crystalline to gel phase transitions. In heterogeneous mixtures such as those found in biological membranes the phase transitions can lead to increases in permeability and lateral phase separations that often are irreparable. Certain sugars are capable of preventing these deleterious events by inhibiting fusion during drying and by maintaining the lipid in a fluid state in the absence of water. As a result, the increased permeability and lateral phase separations that accompany dehydration are absent. The weight of the evidence suggests strongly that there is a direct interaction between the sugars and lipids in the dry state. Although the evidence is less clear about whether these sugars can interact directly with hydrated bilayers, there are strong suggestions in the literature that sugars free in solution or covalently linked to membrane constituents can also affect the physical properties and presumably the stability of bilayers. Finally, we have far less evidence concerning the mechanism by which they do so, but the same sugars are also capable of preserving the structure and function of both membrane-bound and soluble proteins in the absence of water. We believe these effects may be important in the survival of intact cells and organisms such as seeds in the absence of water. Furthermore, in view of the practical importance of preserving biological structures we suspect that the results described here will ultimately have important applications in biology and medicine.

Modes of interaction of cryoprotectants with membrane phospholipids during freezing.

The abilities of a variety of compounds to inhibit liposome fusion during freeze/thaw were assessed by resonance energy transfer. Small unilamellar vesicles have been frozen according to three different protocols. Membrane intermixing was seen to be relatively independent of freezing protocol except when glycerol, dimethyl sulfoxide (DMSO), or sarcosine was used as the cryoprotectant. Low concentrations of polyvinylpyrolidone or 4-hydroxyproline enhanced fusion of liposomes, whereas high concentrations of these compounds had no effect. Glycerol, DMSO, proline, betaine, and sarcosine reduced fusion, but only when their concentrations were greater than 1 M. The most effective cryoprotectants were trehalose and sucrose, which both reduced fusion to minimal levels at concentrations of only 0.2 M. We have also used europium to probe the modes of interaction of these compounds with phospholipids. Europium, which is known to bind to the phosphate headgroup, maximized fusion in liposomes subjected to freeze/thaw. This "europium-induced" fusion was progressively reduced by the presence of increasing sucrose, trehalose, or glycerol, suggesting a competition for the headgroup. However, the presence of proline, betaine, or sarcosine did not reduce europium-induced fusion, suggesting that these compounds do not compete for the headgroup. Substitution of polar side chains on the hydrophobic regions of proline or sarcosine eliminate their cryoprotective properties, suggesting that these compounds interact with the acyl chains of the bilayer.