The freezing point depression of mammalian tissues after sudden heating in boiling distilled water

The calculated freezing point depression of freshly excised boiled mammalian tissue is approximately the same as that of plasma. The boiling procedure was chosen to eliminate the influence of metabolism on the level of the freezing point depression. Problems created by the boiling, such as equilibrium between tissue and diluent, change in activity coefficient by dilution, and loss of CO(2) content, are discussed. A frozen crushed tissue homogenate is hypertonic to plasma. Boiling and dilution of such hypertonic homogenate exposed to room temperature for 5 to 15 minutes did not produce significant or unexplicable decreases in its osmotic activity. Moreover, freezing and crushing of a boiled diluted tissue did not produce any increase of the isoosmotic level of freezing point depression. It is possible to explain these data either with the hypothesis of hypertonic cell fluid or with that of isotonic cell fluid. In the case of an assumed isotonic cell fluid, data can be explained with one assumption, experimentally backed. In the case of an assumed hypertonic theory data can be explained only with the help of at least three ad hoc postulates. The data support the validity of the classical concept which holds that cell fluid is isotonic to extracellular fluid.     

Theoretical and experimental studies on freezing point depression and vapor pressure deficit as methods to measure osmotic pressure of aqueous polyethylene glycol and bovine serum albumin solutions

For survival in adverse environments where there is drought, high salt concentration or low temperature, some plants seem to be able to synthesize biochemical compounds, including proteins, in response to changes in water activity or osmotic pressure. Measurement of the water activity or osmotic pressure of simple aqueous solutions has been based on freezing point depression or vapor pressure deficit. Measurement of the osmotic pressure of plants under water stress has been mainly based on vapor pressure deficit. However, differences have been noted for osmotic pressure values of aqueous polyethylene glycol (PEG) solutions measured by freezing point depression and vapor pressure deficit. For this paper, the physicochemical basis of freezing point depression and vapor pressure deficit were first examined theoretically and then, the osmotic pressure of aqueous ethylene glycol and of PEG solutions were measured by both freezing point depression and vapor pressure deficit in comparison with other aqueous solutions such as NaCl, KCl, CaCl(2), glucose, sucrose, raffinose, and bovine serum albumin (BSA) solutions. The results showed that: (1) freezing point depression and vapor pressure deficit share theoretically the same physicochemical basis; (2) theoretically, they are proportional to the molal concentration of the aqueous solutions to be measured; (3) in practice, the osmotic pressure levels of aqueous NaCl, KCl, CaCl(2), glucose, sucrose, and raffinose solutions increase in proportion to their molal concentrations and there is little inconsistency between those measured by freezing point depression and vapor pressure deficit; (4) the osmotic pressure levels of aqueous ethylene glycol and PEG solutions measured by freezing point depression differed from the values measured by vapor pressure deficit; (5) the osmotic pressure of aqueous BSA solution measured by freezing point depression differed slightly from that measured by vapor pressure deficit.     

Storage of Osmotically Active Compounds in the Taproot of Daucus carota L.

The osmotic potential of cell sap from the storage root, lateralroots and shoots of carrot (Daucus carota L., cv. AmsterdamseBak) was calculated from the concentration of osmotically activecompounds in these tissues. The osmotic potential of the taprootdid not change with age prior to and during the storage of osmoticallyactive sugars, as sucrose and reducing sugars. The increased contribution of soluble sugars in the osmoticpotential was accompanied by a proportionally decreased contributionof potassium and organic acids. Before storage of soluble sugarsin the taproot occurred, potassium and organic acids contributed80% to the total osmotic value, in contrast with lateral roottissue, where potassium and nitrate were the main osmotic solutes.The concentration of osmotically active solutes was lower inlateral root tissue than in storage root tissue. Shoot tissueresembled taproot tissue before storage, in having potassiumand organic acids as the main osmotic solutes. The concentrationof osmotically active solutes was highest in shoot tissue andit increased towards the end of the experimental period. The calculated osmotic potentials were in good agreement withthe experimental values, as determined from the molecular depressionof the freezing point of cell sap. Storage of reducing sugarsand sucrose is discussed in relation to acid and neutral invertaseactivities. Key words: Daucus carota, Osmotic solutes, Sugar storage, Invertase activity     

Efflux of Red Cell Water into Buffered Hypertonic Solutions

Buffered NaCl solutions hypertonic to rabbit serum were prepared and freezing point depressions of each determined after dilution with measured amounts of water. Freezing point depression of these dilutions was a linear function of the amount of water added. One ml. of rabbit red cells was added to each 4 ml. of the hypertonic solutions and after incubation at 38°C. for 30 minutes the mixture was centrifuged and a freezing point depression determined on the supernatant fluid. The amount of water added to the hypertonic solutions by the red cells was calcuated from this freezing point depression. For each decrease in the freezing point of -0.093°C. of the surrounding solution red cells gave up approximately 5 ml. of water per 100 ml. of red cells in the range of -0.560 to -0.930°C. Beyond -0.930°C. the amount of water given up by 100 ml. of red cells fits best a parabolic equation. The maximum of this equation occurred at a freezing point of the hypertonic solution of -2.001°C. at which time the maximum amount of water leaving the red cells would be 39.9 ml. per 100 ml. of red cells. The data suggest that only about 43 per cent of the red cell water is available for exchange into solutions of increasing tonicity.     

OSMOTIC RELATIONSHIPS IN THE HEN'S EGG

Data have been given to illustrate the difficulty of obtaining consistent freezing point data with a viscous fluid such as the yolk of the hen''s egg and a technique has been described for obtaining reproducible and accurate results consistently. Further freezing point data have been given which were obtained with both fertile and unfertile hen''s eggs by the use of a freezing point method previously described by the writer. These data show that there is a pronounced difference between the freezing points of the yolk and the white in contrast to data obtained by the use of the same method by Howard who found the freezing points of the yolk and the white to be the same. It was shown by freezing point determinations that even in a mixture of yolk and white osmotic equilibrium is slowly arrived at. This again emphasizes the fact established by Smith and Shepherd that since osmotic equilibrium between yolk and white is slowly arrived at, the postulation of a vital activity at the yolk membrane is unnecessary, since the steady state previously postulated need not be assumed to exist.     

Examination of some processing methods for freezing boar semen.

Five experiments were conducted to examine the effect of processing methods and diluents on survival and morphology of boar spermatozoa after freezing. Post-thawing survival of spermatozoa was better for Beltsville-F3 (BF3) than for tris-fructose-EDTA freezing diluent when the seminal plasma and glycerol were removed prior to freezing (method A). Both freezing diluents yielded similar viability results when the spermatozoa were frozen in the presence of siminal plasma and glycerol (method B). Viability of spermatozoa after thawing was better when glycerol concentration in the prefreezing diluent (method A) or in the freezing medium (method B) was 2-5 and 5-0 rather than 7-5%. Cooling of diluted semen to 5 degrees C beyond 4 h decreased the post-thawing survival of spermatozoa. The proportion of spermatozoa with undamaged acrosomes after processing and thawing by different methods was indistinguishable and relatively low. When the semen was frozen at cell concentrations ranging from 0-25 to 2-0 X 10(9)/ml, the viability of spermatozoa declined with increasing concentration following freezing in BF3, and S-1 diluents. Viability results were very similar for all cell concentrations examined when tris-fructose-EDTA diluent was used, indicating the possibility of freezing boar semen in a concentrated state.     

Effect of freezing and thawing on the distribution of lysosomal hydrolases in leaves of Solanum tuberosum L.

Density-gradient ultracentrifugation techniques showed that freezing and thawing of potato leaves resulted in a change in the density of subcellular particles containing acid phosphatase and acid ribonuclease (RNase). Gel filtration experiments were used to characterise the molecular forms of the hydrolases associated with the various cell fractions. Freezing and thawing promoted a release of a portion of the complement of acid phosphatase and RNase from the lysosomes to the supernatant fluid fraction of cell homogenates. The freezing treatment appeared to activate latent lysosomal RNase.     

Modelling the effect of osmolality on the bursting strength of yeast cells.

When yeast cells are resuspended in buffer prior to homogenisation, the diluent osmotic pressure can have a significant effect on cell mechanical strength. In this paper a model is proposed which describes the relationship between the cell bursting force and the osmotic pressure of the diluent, using chemical potential and force balance analyses. Yeast cells were exposed for 1 h to diluents with osmolalities varying from almost 0 to 700 mmol kg-1 before their bursting strengths were measured by micromanipulation. The experimental data were compared with the predictions made from the model and in general they were in good agreement. It is expected that this model might be used to understand cell disruption behaviour in downstream processing equipment such as homogenisers.     

Tissue hemolysins as lysin-inhibitor complexes

1. Lytic substances are enzymatically produced at 37°C. from tissue slices or homogenates (mouse liver, kidney, etc.) and appear in the medium in which the tissue fragments are suspended. Their concentration increases with the time during which the tissue is kept at 37°C. (preincubation), and is accompanied by pH changes, so that the lytic activity as finally measured is a function of both the time of preincubation and of the pH. The optimum pH for lysin production is above 7.0, but the lysins, once produced, hemolyze red cells more rapidly at low pH's than at high ones. The enzyme system which produces the lysins is inactivated by heating to 100°C. for 5 minutes. Sodium iodoacetate and fluoride interfere with lysin production principally by reducing the concomitant pH shift; KCN accelerates the production of lytic material in mouse liver homogenates. 2. Comparison of the lytic activity of the supernatant fluid of a preincubated homogenate with the much greater lytic activity of the substances which can be extracted from the same supernatant fluid by alcohol and ether points to these extractable substances existing in the supernatant fluid as lysin-inhibitor complexes of relatively low lytic activity. These complexes are formed enzymatically during preincubation from non-lytic complexes in the tissue. The latter may be lipoproteins, and the highly lytic ether-extractable substances may be fatty acids or their soaps. 3. The diffusibility of the lysin-inhibitor complexes is small. 4. Lytic substances which are ether-insoluble can be extracted with alcohol from tissues as well as from serum. These "lysolecithin-like" substances exist in the supernatant fluids of homogenates as lysin-inhibitor complexes. 5. Lysis of mouse red cells by substances contained in mouse tissue (liver and kidney) is often accompanied by the formation of methemoglobin and choleglobin. Mouse red cells containing choleglobin are abnormally fragile both osmotically and mechanically, and it is possible that a process involving the production of choleglobin, accompanied or followed by globin denaturation, is one which contributes towards the hemolysis which occurs in systems containing tissue slices or homogenates.     

Renal epithelial cell lines (BSC-1, MDCK, LLC-PK1) express 11 beta-hydroxysteroid dehydrogenase activity

Renal tissue of several species has been shown to express considerable 11 beta-hydroxysteroid dehydrogenase (11-HSD, EC 1.1.1.146) activity. However, it is uncertain as to which renal cell types exhibit 11-HSD activity. In the present study, we investigated corticosterone metabolism in BSC-1 cells, a continuous renal epithelial cell line derived from the African green monkey (Cercopithecus aethiops). In incubation experiments using 3H-labelled corticosterone and HPLC, we have demonstrated oxidative 11-HSD activity in intact monolayers of BSC-1 cells as well as in BSC-1 cell homogenates. 11-HSD activity in cell homogenates could be stimulated 7-9-fold by the addition of exogenous NADP+ (1 mM). In contrast, no reductive 11-HSD could be detected either in intact cells or in cell homogenates under various experimental conditions which were designed to favor reductive 11-HSD activity. Pilot experiments were performed in cell homogenates from two other renal epithelial cell lines derived from canine (MDCK) and porcine (LLC-PK1) kidney. They also revealed oxidative but no reductive 11-HSD activity. The data provide evidence for an epithelial localization of renal oxidative 11-HSD activity.     

Effect of a Pulsed Electric Field and Osmotic Treatment on Freezing of Potato Tissue

This work discusses the effects of pulsed electric field (PEF) and osmotic pre-treatments on potato tissue structure and on the freezing and freeze-drying behaviour of this tissue. Potato samples (26-mm diameter, 10-mm height) were treated by PEF (400 V/cm) to high level of disintegration (conductivity disintegration index Z was ≈0.95) and were subjected to osmotic treatment in an aqueous solution of NaCl. The samples were either frozen in an air-blast freezer at air temperature of −80 °C and velocity of 2 m/s or freeze-dried at 0 °C and 0.04-mbar pressure. The scanning electron microscope (SEM) images evidenced similarity in structure of the cell walls and area and morphology of starch granules for untreated and PEF-treated potato tissues. However, sequential (PEF + osmotic) pre-treatment of potato tissue resulted in starch granules with rougher surface. The profiles of freezing curves were strongly dependent on pre-treatment. The longest effective freezing time t _f was observed for untreated tissue, and the values of t _f were decreasing in the following sequence: untreated > PEF pre-treated > PEF + osmotically pre-treated. The faster freezing and freeze drying and visually better quality of the dried samples were observed for PEF or sequential PEF + osmotic pre-treatments. The SEM analysis revealed also a noticeable disorder of starch granule surface morphology inside the cells of the freeze-dried potatoes after sequential PEF + osmotic pre-treatment.     

Influence of smear preparation and fixatives on the DNA ploidy and the morphonuclear features of the MXT mammary tumor and normal tissues in the mouse

We compared cytomorphonuclear parameters--related to DNA histogram and chromatin distribution--on MXT mouse mammary tumor and murine normal cells from fresh squash smears or from deparaffinized tissue smears fixed in several fluids. We used the SAMBA 200 cell image processor with software allowing for the discrimination of parameters computed on Feulgen-stained nuclei. The spectrophotometric results--assessed by integrated optical density values--indicate that the nuclei from deparaffinized tissue fixed in Bouin's fluid are around 50% less stained than those fixed in formalin or ethanol-formalin-acetic acid (EFA). The fresh smears of nuclei fixed in formalin contain a less well-defined and more homogeneous chromatin than after Bouin's or EFA fixation. This has led to the conclusion that morphonuclear parameter comparisons performed on tissues differently processed or from different origins present severe limitations.     

Mechanisms of intracellular ice formation.

The phenomenon of intracellular freezing in cells was investigated by designing experiments with cultured mouse fibroblasts on a cryomicroscope to critically assess the current hypotheses describing the genesis of intracellular ice: (a) intracellular freezing is a result of critical undercooling; (b) the cytoplasm is nucleated through aqueous pores in the plasma membrane; and (c) intracellular freezing is a result of membrane damage caused by electrical transients at the ice interface. The experimental data did not support any of these theories, but was consistent with the hypothesis that the plasma membrane is damaged at a critical gradient in osmotic pressure across the membrane, and intracellular freezing occurs as a result of this damage. An implication of this hypothesis is that mathematical models can be used to design protocols to avoid damaging gradients in osmotic pressure, allowing new approaches to the preservation of cells, tissues, and organs by rapid cooling.     

Distinction between the G0 and the late G1 phase of rat cells in vivo and in vitro with the nuclear QDH-fluorescence pattern

The quinacrine dihydrochloride (QDH) staining and the [3H]thymidine incorporation patterns were simultaneously analyzed in nuclei of rat cells from a proliferating (granulation tissue) and a nonproliferating tissue (liver). Nuclei from freshly isolated and cultured cells of the rapidly proliferating subcutaneous granulation tissue showed a cell cycle-related pattern similar to that previously described with growing fibroblast-like cells in vitro. Nuclei of liver cells in smears from biopsies and in histological sections showed a fluorescence pattern similar to that of serum-deprived arrested G0 cells from established cell lines. Treatment of primary cultured rat hepatocytes with phenobarbital altered their degree of chromatin condensation similar to that seen after treatment of rats in vivo. The data indicate that the QDH staining pattern is an early marker, suitable for detecting the cell cycle-promoting activity of chemicals (e.g., of tumor promoters) in nonproliferating cells from various tissues in vivo and in vitro.     

The plant cell pressure probe

The pressure probe is a micro manometer for the simultaneous direct recording and manipulation of plant cell hydrostatic pressure. It is used to map in space and time the turgor pressures of individual cells within tissues and organs of intact plants. This is used to study the hydraulic architecture of tissues, tissue movement and the responses of tissues to water stress. The approach can be augmented by simultaneous measurement of individual cell osmotic pressure. This permits the hydraulic driving forces across selectively permeable membranes and walls to be assessed fully. By manipulating manually the pressure, cell wall elasticity and its properties can also be mapped. Under some conditions this can be extended to plastic behaviour.     

On the osmotically unresponsive water compartment in cells

Differences in colligative properties (freezing point, boiling point, vapor pressure and osmotic behavior) between water in living cells and pure bulk water were investigated by re-evaluating reports of the osmotic behavior of mammalian cells. In five different animal cells, osmotically unresponsive water (OUW) values ranged from 1.1 to 2.2 g per g dry mass. Detailed analysis of human red blood cell (RBC) data indicates a major role for hemoglobin OUW-values, aggregation and packing in cell volume regulation that can be explained for the first time in relevant molecular terms.     

Cryopreservation of cartilage cell and tissue for biobanking

Preservation of cell and tissue samples from endangered species is a part of biodiversity conservation strategy. Therefore, setting up proper cell and tissue cryopreservation methods is very important as these tissue samples and cells could be used to reintroduce the lost genes into the breeding pool by nuclear transfer. In this study, we investigated the effect of vitrification and slow freezing on cartilage cell and tissue viability for biobanking. Firstly, primary adult cartilage cells (ACCs) and fetal cartilage cells (FCC) were cryopreserved by vitrification and slow freezing. Cells were vitrified after a two-step equilibration in a solution composed of ethylene glycol (EG), Ficoll and sucrose. For slow freezing three different cooling rates (0.5, 1 and 2 °C/min) were tested in straws. Secondly, the tissues taken from articular cartilage were cryopreserved by vitrification and slow freezing (1 °C/min). The results revealed no significant difference between the viability ratios, proliferative activity and GAG synthesis of cartilage cells which were cryopreserved by using vitrification or slow freezing methods. Despite the significant decrease in the viability ratio of freeze–thawed cartilage tissues, cryopreservation did not prevent the establishment of primary cell cultures from cartilage tissues. The results revealed that the vitrification method could be recommended to cryopreserve cartilage tissue and cells from bovine to be used as alternative cell donor sources in nuclear transfer studies for biobanking as a part of biodiversity conservation strategy. Moreover, cartilage cell suspensions were successfully cryopreserved in straws by using a controlled-rate freezing machine in the present study.     

The potential of water in mammalian tissues

Melting point depression was used as an index of the water potential of rat tissues and serum. Organs removed from anesthetized rats were immediately frozen in liquid nitrogen and ground with mortar and pestle. Aliquots of the resulting frozen powder were suspended in chilled liquid silicone. While the suspension was vigorously stirred and warmed at a constant rate, the temperature of the melting mixture was measured. The melting curves of rat muscle, liver, heart, and brain were not significantly different from those of rat serum. The melting curve depression of whole kidney was greater than that of serum; this was demonstrated to be due to hypertonicity of the renal medullary area alone. It was demonstrated that autolysis will rapidly increase the depression of the melting curve of tissue. It is concluded that within the limits of the method used the melting point depression, and hence the water potential, of intracellular and extracellular fluids is the same.     

Osmotic properties of pea internodes in relation to growth and auxin action

The water transport properties of etiolated pea (Pisum sativum L.) internodes were studied using both dynamic and steady-state methods to determine (a) whether water transport through the growing tissue limits the rate of cell enlargement, and (b) whether auxin stimulates growth in part by increasing the hydraulic conductance of the growing tissue.

Measurements using the pressure probe technique showed that the hydraulic conductivity of cortical cell membranes was the same for both slowly growing and auxin-induced rapidly growing cells (membrane hydraulic conductivity, about 1.5 × 10⁻⁵ centimeters per second per bar). In a second technique which measured the rate of water movement through the entire pea internode, the half-time for radial water flow was about 60 seconds and was not altered by auxin application. These results indicate that auxin does not alter the hydraulic conductance of pea stem tissue, either at the cellular or the whole tissue level.

Measurements of the turgor pressure of cortical cells, combined with osmotic pressure measurements of expressed cell sap, show that the water potential of growing pea stems was about −3 bars. When the growth rate was altered by various treatments, including decapitation, auxin application, cold temperature, and KCN treatment, the water potential was independent of the growth rate of the stem. We attribute the depression of the water potential in young pea stems to the presence of solutes in the cell wall free space of the tissue. This interpretation is supported by the results of infiltration and perfusion experiments.

From the results of these dynamic and steady-state experiments, we conclude that the internal gradient in water potential (from the xylem to the epidermis) needed to sustain cell enlargement is small (no greater than 0.5 bar). Thus, the hydraulic conductance of the tissue is sufficiently large that it does not control or limit the rate of cell enlargement.

     

Tobacco mosaic virus infection stimulates the phosphorylation of a plant protein associated with double-stranded RNA-dependent protein kinase activity

The influence of tobacco mosaic virus (TMV) infection on nucleotide binding and phosphorylation of an Mr 68,000 host-encoded protein (p68) was examined. The phosphorylation of p68 in homogenates from TMV-infected tissues was 4-fold greater than in homogenates from mock inoculated tissues. Phosphorylation of p68 in extracts from mock inoculated tissues was enhanced by the addition of double-stranded (ds) RNA. Nucleotide photoaffinity labeling experiments indicate that p68 contains an ATP binding site with characteristics consistent with protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase activity. Antiserum raised against a dsRNA-dependent protein kinase from interferon-treated human cells immunoprecipitated p68 from extracts of TMV-infected tissue, and p68-containing immunocomplexes catalyzed the phosphorylation of endogenous p68. These data suggest that p68 may be an autophosphorylating, dsRNA-dependent protein kinase involved in viral pathogenesis. Based upon analogous functions demonstrated for dsRNA-dependent protein kinases in mammalian systems, p68 may have a role in the regulation of protein synthesis and viral replication in infected cells.