Analysis of the water permeability of human granulocytes at subzero temperatures in the presence of extracellular ice

The plasma membrane water permeability of human granulocytes in the presence of extracellular ice was determined experimentally on a cryomicroscope. Transient volumes of individual cells were measured at constant subzero temperatures subsequent to ice nucleation. Permeability values were deduced by adjustment of multiple parameters in a model to obtain an optimal fit to the data. The permeability was determined to be a function of both temperature and intracellular solute osmolality, with a reference value at 0 degrees C of 0.407 micrometers/atm.min and temperature and solute coefficients of 218kJ/mol and 1.09 Osm/kg.     

Determination of the water permeability (Lp) of mouse oocytes at −25 °C and its activation energy at subzero temperatures

Typically, subzero permeability measurements are experimentally difficult and infrequently reported. Here we report an approach we have applied to mouse oocytes. Interrupted cooling involves rapidly cooling oocytes (50 °C/min) to an intermediate temperature above the intracellular nucleation zone, holding them for up to 40 min while they dehydrate, and then rapidly cooling them to −70 °C or below. If the intermediate holding temperature and holding time are well chosen, high post thaw survival of the oocytes is possible because the freezable water is removed during the hold. The length of time required for the exit of the freezable water allows the water permeability at that temperature to be determined. These experiments used 1.5 M ethylene glycol in PBS and included a transient hold of 2 min for equilibration at −10 °C, just below the extracellar ice formation temperature. We obtain an Lp = 1.8 × 10⁻³ μm min⁻¹ atm⁻¹ at −25 °C based on a hold time of 30 min yielding 80% survival and the premise that most of the freezable water is removed during the 30 min hold. If we assume that the water permeability is a continuous function of temperature and that its Ea changes at 0 °C, we obtain a subzero Ea of 21 kcal/mol; higher than the suprazero value of 14 kcal/mol. A number of assumptions are required for these water loss calculations and the resulting value of Lp can vary by up to a factor of 2, depending on the choices make.     

Stopped flow method at subzero temperatures

A stopped-flow apparatus especially adapted for experiments with aqueous-organic solutions of enzymes at subzero temperatures is described.Performance data are given and discussed in a study of the reaction between ascorbate and 2,6-dichlorophenol-indophenol (DCPIP) in an “antifreeze” aqueous-organic mixture as a function of temperature.     

Isoelectric focusing and electrophoresis at subzero temperatures

Electrophoretic and isoelectric focusing separations have been achieved at ?20 to ?30°C, i.e., at temperatures considerably lower than previously reported by using as supporting media gels of acrylamide-methylacrylate copolymers and dimenthylsulfoxide-water mixtures. Hybrids of human and sickle cell hemoglobin and partially oxidized human carboxyhemoglobin have been separated in the temperature range ?20 to ?30°C, both by a discontinuous buffer gel electrophoresis and by isoelectric focusing.     

Freeze-fixation at high subzero temperatures.

Previous attempts to determine the distribution of ice in frozen tissues at high sub-zero temperatures generally called for the further cooling of the tissues in question to facilitate freeze-drying, freeze-substitution, and freeze-fracture replication. Direct cryomicroscopic determinations, free from uncertainties stemming from changes in sample temperature could, it seemed, only be made in certain special cases. We have presented an isothermal “freeze-fixation” procedure designed to permit, instead, the postthaw retention of the freezing pattern and the conventional processing, afterward, of the thawed specimen. The method demands the exposure of the frozen tissues to fixative solutions incapable of dissolving ice. Frozen specimens are immersed in aqueous fixative solutions prepared in each instance (1) to freeze at a temperature equal to that at which fixation is to be conducted, (2) to contain quantities of finely divided ice sufficient to guarantee the maintenance of a constant water activity. Frozen frog and rat hearts and skeletal muscle tissues were exposed to formaldehyde, formaldehyde/ glutaraldehyde, and glutaraldehyde solutions at ?2, ?5, and ?10 °C, the temperatures being maintained in each case to ± 0.1 °C, or better. Tissues withdrawn at intervals were thawed, postfixed, dehydrated, embedded, and sectioned. The sections demonstrated the retention, after thawing, of structural features characteristic of the frozen state. The small hearts we exposed to formaldehyde were fixed throughout in 3 hr at ?2 ° and in 20 hr at ?5 °C. The action of osmium tetroxide was investigated. The method appears to be well-suited to numerous experimental applications.     

Glycerol permeability of human spermatozoa and its activation energy

Glycerol has commonly been employed as a cryoprotectant in cryopreservation of human spermatozoa. However, the addition of glycerol into the sperm before freezing and the removal of glycerol from the sperm after freezing and thawing result in anisotonic environments to the cells, which can cause cell injury. To define optimal procedures for the addition/removal of glycerol and to minimize the cell injury, one needs to know the kinetics of glycerol permeation across the sperm plasma membrane at different temperatures. For this, one has to determine the permeability coefficient of glycerol (Pg) and its activation energy (Ea). Values of Pg at different temperatures and at different glycerol concentrations were determined by measuring the time required for 50% spermolysis in hyperosmotic glycerol solutions which were hypotonic with respect to electrolytes. Value of the Ea was determined assuming an Arrhenius type temperature dependence of Pg. A dual fluorescent staining technique (propidium iodide and 6-carboxyfluoroscein diacetate) and flow cytometry were used to measure the spermolysis. The values of Pg in 0.5, 1.0, 1.5, and 2.0 M glycerol at 22 degrees C are 1.62, 1.88, 1.68, and 1.54 x 10(-3) cm/min, respectively. The values of Pg in 1 M glycerol at 0, 8, 22, and 30 degrees C are 0.33, 0.54, 1.88, and 2.60 x 10(-3) cm/min, respectively. The value of Ea is 11.76 kcal/mol.     

Cytochrome c-cytochrome oxidase interaction at subzero temperatures.

Cytochrome oxidase forms two distinctive compounds with oxygen at --105 and --90 degrees C, one appears to be oxycytochrome oxidase (Compound A) and the other peroxycytochrome oxidase (Compound B). The functional role of compound B in the oxidation of cytochrome c has been examined in a variety of mitochondrial preparations. The rate and the extent of the reaction have been found to be dependent upon the presence of a fluid phase in the vicinity of the site of the reaction of cytochrome c and cytochrome oxidase. The kinetics of cytochrome c oxidation and of the slowly reacting component of cytochrome oxidase are found to be linked to one another even in cytochrome c depleted preparations, but under appropriate conditions, especially low temperatures, the oxidation of cytochrome c precedes that of this component of cytochrome oxidase. Based upon the identification of the slowly reacting components of cytochrome oxidase with cytochrome c, various mechanisms are considered which allow cytochrome c to be oxidized without the intervention of cytochrome a at very low temperatures, and tunneling seems an appropriate mechanism.     

Bacterial genome replication at subzero temperatures in permafrost

Microbial metabolic activity occurs at subzero temperatures in permafrost, an environment representing ∼25% of the global soil organic matter. Although much of the observed subzero microbial activity may be due to basal metabolism or macromolecular repair, there is also ample evidence for cellular growth. Unfortunately, most metabolic measurements or culture-based laboratory experiments cannot elucidate the specific microorganisms responsible for metabolic activities in native permafrost, nor, can bulk approaches determine whether different members of the microbial community modulate their responses as a function of changing subzero temperatures. Here, we report on the use of stable isotope probing with ¹³C-acetate to demonstrate bacterial genome replication in Alaskan permafrost at temperatures of 0 to −20 °C. We found that the majority (80%) of operational taxonomic units detected in permafrost microcosms were active and could synthesize ¹³C-labeled DNA when supplemented with ¹³C-acetate at temperatures of 0 to −20 °C during a 6-month incubation. The data indicated that some members of the bacterial community were active across all of the experimental temperatures, whereas many others only synthesized DNA within a narrow subzero temperature range. Phylogenetic analysis of ¹³C-labeled 16S rRNA genes revealed that the subzero active bacteria were members of the Acidobacteria, Actinobacteria, Chloroflexi, Gemmatimonadetes and Proteobacteria phyla and were distantly related to currently cultivated psychrophiles. These results imply that small subzero temperature changes may lead to changes in the active microbial community, which could have consequences for biogeochemical cycling in permanently frozen systems.     

paH gradients in isoelectric focusing experiments at subzero temperatures

We have developed a simple enzymatic procedure for evaluation of antisera reactivity against the large molecular forms of gastrin and cholecystokinin (CCK). The procedure can be used for radioimmunochemical quantitation of the precursor molecules. The different molecular forms of gastrin or CCK in tissue extracts or plasma were separated by gel chromatography. The concentration of each form was then measured with 17 different antisera before and after tryptic cleavage. The ratio between the molar concentrations before and after tryptic cleavage varied from 0.32 to 1.00. Such variation can explain the variable hormone concentrations in serum and tissue measured with different radioimmunoassays. The present procedure can be performed with any biological fluid containing the precursor forms. It does not require the large molecular forms in pure state. In principle the procedure can be used for quantitation of all peptide precursors.     

High-pressure inactivation of Saccharomyces cerevisiae and Lactobacillus plantarum at subzero temperatures

High hydrostatic pressure is a new technology in the food processing industry, and is used for cold pasteurization of food products. However, the pressure inactivation of food-borne microorganisms requires very high pressures (generally more than 400 MPa) and long pressure holding times (5 min or more). Carrying out pressure processing at low temperatures without freezing can reduce these parameters, which presently limit the application of this technology, in keeping the quality of fresh raw product. The yeast, Saccharomyces cerevisiae and the bacterium, Lactobacillus plantarum were pressurized for 10 min at temperatures between -20 and 25 degrees C and pressure between 100 and 350 MPa. Pressurization at subzero temperatures without freezing significantly enhanced the effect of pressure. For example, at a pressure of 150 MPa, the decrease in temperature from ambient to -20 degrees C allowed an increase in the pressure-induced inactivation from less than 1 log up to 7-8 log for each microorganism studied. However, for comparable inactivation levels, the kinetics of microorganism inactivation did not differ, which suggests identical inactivation mechanisms. Implications of water thermodynamical properties like compression, protein denaturation, as well as membrane phase transitions, are discussed.     

Cytochrome c-cytochrome oxidase interaction at subzero temperatures

Cytochrome oxidase forms two distinctive compounds with oxygen at ?105 and ?90°C, one appears to be oxycytochrome oxidase (Compound A) and the other peroxycytochrome oxidase (Compound B). The functional role of compound B in the oxidation of cytochrome c has been examined in a variety of mitochondrial preparations. The rate and the extent of the reaction have been found to be dependent upon the presence of a fluid phase in the vicinity of the site of the reaction of cytochrome c and cytochrome oxidase. The kinetics of cytochrome c oxidation and of the slowly reacting component of cytochrome oxidase are found to be linked to one another even in cytochrome c depleted preparations, but under appropriate conditions, especially low temperatures, the oxidation of cytochrome c precedes that of this component of cytochrome oxidase. Based upon the identification of the slowly reacting components of cytochrome oxidase with cytochrome c, various mechanisms are considered which allow cytochrome c to be oxidized without the intervention of cytochrome a at very low temperatures, and tunneling seems an appropriate mechanism.     

Kinetics of water loss from cells at subzero centigrade temperatures

A mathematical model is developed for the calculation of the kinetics of water loss from cells at subzero centigrade temperatures. In this model it is assumed that the cell surface membrane is permeable to water only, the protoplasm is a nonideal solution, the cells are spherical, and during the cooling process the cell temperature is not uniform inside the cell. It is also assumed that because of water loss due to cooling process the cell volume and the cell surface area reduce and the reductions in surface area and volume of the cell are functions of the amount of water loss from the cell. Based on this model, and for different conditions, the fractions of supercooled intracellular water remaining in the cells at various temperatures are calculated.It is shown that for cooling cells at subzero centigrade temperatures. (1) the consideration of Clausius-Clapeyron equation for vapor pressures of water and ice, instead of the exact vapor pressure relations, may produce errors in the prediction of the amount of water loss from the cells at high cooling rates only, (2) the assumption of intact cells will produce considerable deviation in the prediction of water loss from the cells as compared to the more realistic assumption of shrinkable cells, (3) the nonideality of protoplasm solution is very effective on the prediction of the amount of water loss from the cells, and (4) the assumption of uniform-temperature cells during the cooling process may be erroneous only for cells with small fractions of water in their protoplasms.     

Freezing preservation of adult mammalian heart at high subzero temperatures

The present study adapted the overwintering strategy employed by freeze-tolerant amphibians and reptiles to freeze-preserve the isolated rat heart. The heart was flushed with a cardioplegic solution and supercooled to -1.2 and -3 degrees C. Then freezing was induced by inoculation of ice crystal. The viability of the heart explant was assessed after reanimation by the isolated working heart perfusion. There was no recovery of function in hearts flushed with solution containing 0.28 mM CaCl2. Lowering the concentration of CaCl2 to 0.15 mM, however, rendered good functional return. Furthermore, inclusion of 50 mM glycerol in the flush solution dramatically improved functional preservation. Under the best conditions defined here, the recoveries of aortic flow, coronary flow, cardiac output, systolic pressure, and work in hearts stored at -1.2 degrees C for 3 h were 72.8 +/- 6.8, 87.2 +/- 4.2, 77.6 +/- 5.4, 83.4 +/- 2.8, and 66.6 +/- 5.9% (mean +/- SEM, n = 8) of the unstored control levels, respectively. The myocardial ice content was 18.6 +/- 5.4% (n = 5) of tissue water. Prolonging the storage time to 5 h increased the ice content to 45.3 +/- 8.1% and reduced the recovery of cardiac output to 23 +/- 11% of the control value (mean +/- SEM, n = 5). Hearts frozen at -3 degrees C for 1.5 h showed 29.4 +/- 8.7% (n = 3) of control cardiac output during reperfusion. This novel approach may provide an opportunity to advance our knowledge about freezing preservation of not only the heart but other solid organs as well.     

Motility of Colwellia psychrerythraea strain 34H at subzero temperatures

We examined the Arctic bacterium Colwellia psychrerythraea strain 34H for motility at temperatures from -1 to -15 degrees C by using transmitted-light microscopy in a temperature-controlled laboratory. The results, showing motility to -10 degrees C, indicate much lower temperatures to be permissive of motility than previously reported (5 degrees C), with implications for microbial activity in frozen environments.