The contribution of the cryoprotectant to total injury in rabbit hearts frozen with ethylene glycol.

Following the failure of hearts to recover function after freezing at ?20 ° in the presence of 3 m ethylene glycol, a variety of experimental treatments was devised to determine the relative harmfulness of ice, high concentrations of electrolytes and high ethylene glycol concentration. Neither cooling to ?20 °C without freezing in a Ca²⁺-free solution containing twice the normal salt concentration and 6 m ethylene glycol (freezing 3 m ethylene glycol at ?20 °C doubles the solute concentration in the liquid phase), nor perfusion at ?1 °C with this solution were conducive to the recovery of hearts. However, perfusion with Ca²⁺-free 3 m ethylene glycol solution with twice the normal concentration of salts did allow full recovery of function, whereas perfusion with Ca²⁺-free 6 m ethylene glycol solution with normal salt concentrations did not. Therefore, the high ethylene glycol concentration encountered during freezing was the main cause of damage.     

Cryoprotective effect of methanol during cooling of frog hearts

It has been shown that frog hearts, perfused with gradually increasing concentrations of ethylene glycol (to 11 m) as the temperature was gradually lowered to ?55 °C and then cooled abruptly to ?78 °C, resumed spontaneous contractions when rewarmed. The thin-walled sinus venosus and atria showed significantly better recovery than the thick-walled ventricle. It was suggested that the difference in recovery of the various parts of the heart might be related to the degree of penetration of the glycol into the tissue. In an attempt to achieve better penetration during perfusion, in particular at subzero temperatures, methanol was substituted for glycol in the perfusate. Hearts equilibrated at room temperature in nontoxic concentrations of methanol were perfused with gradually increasing concentrations as the specimen was gradually cooled to various temperatures. The hearts were gradually rewarmed, and during the rewarming the concentrations of methanol in the perfusate was gradually reduced. All hearts resumed spontaneous rhythmic contractions providing they were not cooled to below ?30 °C or perfused with methanol solutions exceeding 10 m concentration. Cooling to lower temperatures and exposure to higher concentrations of methanol did not permit recovery. These results show that at temperatures as low as ?30 °C methanol in concentrations up to 10 m is comparable to ethylene glycol in its ability to protect hearts from cryoinjury. Its failure to protect at lower temperatures may be related to the development of toxic concentrations when water is removed in the form of ice.     

The response of the glass/calomel pH-cell in aqueous solutions containing dimethylsulphoxide at 25 and-12 degrees C.

The pH response of the glass/calomel electrochemical cell in aqueous solutions containing dimethylsulphoxide at 25 and ?12 °C has been assessed using buffer solutions whose standard pH values were assigned using the hydrogen-silver, silver chloride primary standardising cell. The glass/calomel pH cell was shown to function normally in 30% DMSO-H₂O at 25 and ?12 °C and its response, compared to the hydrogen electrode, was as good as its response in the standard aqueous system at room temperature.     

Water Crystallisation of Model Sugar Solutions with Nanobubbles Produced from Dissolved Carbon Dioxide

This study was conducted to examine the influence of CO₂ nanobubbles on crystallisation behaviour of water during freezing of model sugar (2–5%w/v) solutions. CO₂ gas was dissolved at 0, 1000, and 2000-ppm concentrations before freezing. Carbonated sugar solutions in 50 mL plastic tubes were immersed in a pre-cooled (−15 °C) ethylene glycol bath and left to freeze at −15 °C for 90 min. When the temperature of the solutions reached 0 °C, ultrasound (US; 20 kHz) was emitted in the bath for 20 s duration through a metal horn transducer. The US wave applied in the ethylene glycol bath was expected to propagate to the sugar solutions in the tube and promote gas bubble formation from dissolved CO₂, which will trigger the ice nucleation. Obtained freezing curves were analysed for nucleation time and temperature, supercooling degree, and time taken for phase change. In general, the CO₂ gas promoted freezing of water, causing a noticeable shift in nucleation parameters. For example, nucleation time of 2000-ppm carbonated water coupled with sonication emission for 20 s (7.8 min) was much shorter than that of controls (pure water without any treatment = 19.1 min and US only = 14.3 min). The former initiated ice nucleation just below sub-zero temperature (−0.2 °C) whereas the onset temperature of controls (pure water without any treatment = −11.3 °C and the US only treatment = −10.3 °C). A similar effect was observed with different model sugar solutions. The current findings can be applied to refine the manufacturing process of ice-cream and frozen desserts by the food industries.

     

Microwave properties of cryoprotectants

The values of the dielectric constant and of the loss tangent for pure samples of DMSO, ethylene glycol, and glycerol were determined over the temperature range of + 15 to −70 °C. An operating frequency range of 1.45 to 1.55 GHz was used, allowing direct application of the results of both 0.915 and 2.450 GHz studies. Strong temperature dependencies were found, with peaks and irregularities occuring at subfreezing temperatures. In order to design a suitable cryoprotective system for the long term preservation of whole organs, the effect of cryoprotectant concentration on microwave properties must be known.     

An Efficient Method for Separating Ascospores from Sporulating Cultures in Saccharomyces cerevisiae by Hydrostatic Pressure

A new oxidative reaction of ethylene glycol was found with two alcohol oxidases from methanol yeast, Candida sp. and Pichia pastoris. Both alcohol oxidases oxidized ethylene glycol to glyoxal via glycolaldehyde. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Candida alcohol oxidase were around 8.5 and 5.5, respectively, and their apparent K_ms were 2.96 m and 28.6 mm, respectively. The optimum temperature was 40°C at pH 7.0. The optimum pHs for the oxidation of ethylene glycol and glycolaldehyde by the Pichia alcohol oxidase were around 8.0 and 6.0, respectively, and their optimum temperatures were 50 and 45°C, respectively, at pH 7.0. The apparent K_m for glycolaldehyde was found to be 83.3 mm. For the accumulation of glyoxal, addition of catalase was effective, and a higher amount of glyoxal was obtained at a much lower temperature than the optimum for the alcohol oxidase. When 0.1 m ethylene glycol and glycolaldehyde were incubated with 80 units of the Pichia enzyme at 10°C, both substrates were almost completely converted to glyoxal after 10 and 3h of incubation, respectively.     

Transplantation of rabbit kidneys perfused with glycerol solutions at 10 degrees C.

Experiments are described in which rabbit kidneys were perfused with solutions containing 2, 3, or 4 m glycerol and then were autografted in order to measure any functional impairment. The basic perfusate contained Haemaccel (17.5 g/litre) and mannitol (111 mm) and had a total osmolality of 400 mosmol/kg. The perfusion temperature was 10 °C. Glycerol concentration was slowly increased at a rate of 30 mm min^?1 to 2, 3, or 4 m, was held at the maximum concentration for 30 min, and was then decreased to < 0.1 m at 30 mm min^?1. An immediate contralateral nephrectomy was carried out, and the function of the perfused kidney was assessed by serial measurements of blood urea and serum creatinine, by the determination of 24-hr endogenous creatinine clearance at 1 month and by histological examination. It was found that a concentration of 3 m glycerol was readily tolerated.     

Effect of cryoprotectors on the structural state of liposomes cooled to minus 25 degrees C

Cryoprotectors (propylene glycol), ethylene glycol, polyethylene glycol-1500 and dimethyl sulphoxide) are studied for their effect on permeability of liposomes for incorporated molecules of 5,5-dithiobis-2-nitrobenzoic acid (DTNB) under cooling within a temperature range from 0 degree C to -25 degrees C. A similarity is found in the way of ethylene glycol and propylene glycol, dimethyl sulphoxide and polyethylene glycol-1500 effect on the liposome permeability way. Cooling in the presence of ethylene glycol and propylene glycol causes changes in liposome permeability with a local maximum at -18 degrees C. In the medium with 2M NaCl and ethylene glycol, liposomes were resistant to cooling. Dimethyl sulphoxide and polyethylene glycol-1500 induced a two-phase kinetics of changes in liposome permeability, the first phase being within the 0 = -9 degrees C and the second--within -9--25 degrees C temperature ranges. The found differences are supposed to be associated with the effect of the cryoprotective compounds on the lipid crystallization in a lower-temperatures range.     

Winter survival of an adult bark beetle Ips acuminatus Gyll

Freezing-susceptible adult Ips acuminatus hibernate underneath bark of Scots pine. The beetles lower their supercooling points from ?20 to ?34°C due to accumulation of low molecular weight antifreezes. The capability of specimens to supercool to about ?20°C in the absence of cryoprotective solutes during winter, seemed to be at least partially attributable to the presence of a thermal hysteresis factor at 3–4°C.Using a GC-MS-COM technique, a unique combination of accumulated solutes present only in specimens demonstrating supercooling points below ?20°C was identified as ethylene glycol, mannitol, sorbitol and dulcitol. Not previously found in nature, ethylene glycol was the major solute (90%) synthesized at sub-zero temperatures. Exposure to ?10°C was an effective cue to accumulation of ethylene glycol and nearly 5 times as effective in promoting sorbitol synthesis than was ?5°C. When low molecular weight substances were lost at high temperatures, they were not re-synthesized in beetles re-exposed to sub-zero temperature. The supercooling point was closely related to both the concentration of ethylene glycol and to the haemolymph melting point. Attempts to correlate changes in sorbitol concentrations to changes in supercooling points were not conclusive.Proliferation of thermal hysteresis was observed in the beginning of November. A melting-hysteresis freezing point differential of about 3.6°C was demonstrated in the haemolymph of beetles during December. No thermal hysteresis was demonstrated in the haemolymph of positive phototactic beetles or in the outdoor beetles in May. The combination of high temperature and long photoperiod appeared to be a more effective cue to the final loss of thermal hysteresis than was high temperature alone.     

Function of rabbit kidneys in vitro at normothermia following equilibration with 3.0 M Me2SO and removal by hypertonic washout at 10 degrees C

In the present study, rabbit kidneys were assayed for function on a 37 °C in vitro perfusion system after perfusion on a 10 °C perfusion system which permits the slow introduction and removal of cryoprotectant. The final concentration of 3.0 M Me₂SO was introduced slowly at two different rates. The washout was achieved by perfusion with Me₂SO-free solutions made hypertonic with mannitol. Two regimens of washout were used: 800, 700, 600, 500, and 400 mOsm/kg; and 600, 500, and 400 mOsm/kg.During perfusion at 37 °C, the glomerular filtration rate was similar in all groups and this increased significantly in all groups with time. Protein leakage was minimal. All three Me₂SO groups showed a depressed Na reabsorption capacity, but the 800 mOsm group was the most severely affected. This was also found with glucose reabsorption. We concluded that rabbit kidneys will function well with the cryoprotectant Me₂SO up to 3 M concentration when introduced slowly and washed out with hypertonic mannitol beginning at 600 mOsm/kg. When 800 mOsm is used at the initial step, the proximal tubular function is severely affected.     

The pH-dependent recovery of smooth muscle from storage at -13 degrees C in unfrozen media.

The function of isolated strips of smooth muscle from the guinea pig were assessed by isometric contractile responses to histamine before and after storage at ?13 °C in high potassium, Pipes-buffered solutions containing 30% (w/v) Me₂SO and having different pH values. The structural integrity of the tissue during and after cooling was also assessed by electron microscopy.The pH-dependence of tissue recovery in these muscles was clearly demonstrated after about 17-hr storage at ?13 °C. Despite the wide difference in functional recoveries between groups of muscles cooled in either “high” (7.7) or “low pH” (6.4) media, however, there were no apparent ultrastructural differences between the two differently treated groups of muscles. Muscles from both pH groups, fixed after rewarming to 37 °C, showed the morphological characteristics typically seen in noncooled muscles. An examination of muscle cells which were fixed at ?13 °C (w/v) Me₂SO revealed that the contractile myofibrils of specimens in both experimental groups had undergone a noticeable reorganization and aggregation.     

Impact of In-Situ CO2 Nano-Bubbles Generation on Freezing Parameters of Selected Liquid Foods

The impact of in-situ CO₂ nano-bubbles generation on the freezing properties of soft serve, milk, and apple juice was investigated. Carbonated (0, 1000, and 2000 ppm) liquid foods contained in a tube were submerged and cooled for 90 min in a pre-set ethylene glycol bath (−15 °C). Before the enclosed liquid reached 0 °C, the vibration was discharged through ultrasound in the bath to create nano-bubbles within the carbonated food samples, and the changes in temperature for 90 min of each food were recorded as a freezing curve. The time for onset of nucleation of control soft serve mix was halved in samples with 2000-ppm CO₂ due to the presence of nano-bubbles. Likewise, the nucleation time for milk with and without nano-bubbles at the same CO₂ concentration of 2000 ppm was 7.9 ± 0.1 and 2.8 ± 0.8 min_, respectively. The generation of CO₂ nano-bubbles from 2000-ppm CO₂ level in 10 ^oBx apple juice displayed −9.3 ± 0.3 °C nucleation temperature while the control one had −11.7 ± 0.9 °C.

     

Enzyme activity down to -100 degrees C

The activities of two enzymes, beef liver catalase (EC 1.11.1.6) and calf intestine alkaline phosphatase (EC 3.1.3.1), have been measured down to -97 degrees C and -100 degrees C, respectively. Enzyme activity has not previously been measured at such low temperatures. For catalase, the cryosolvents used were methanol:ethylene glycol:water (70:10:20) and DMSO:ethylene glycol:water (60:20:20). For alkaline phosphatase, methanol:ethylene glycol:water (70:10:20) was used. All of the Arrhenius plots were linear over the whole of the temperature range examined. Since the lowest temperatures at which activity was measured are well below the dynamic transition observed for proteins, the results indicate that the motions which cease below the dynamic transition are not essential for enzyme activity. In all cases the use of cryosolvent led to substantial increases in Arrhenius activation energies, and this imposed practical limitations on the measurement of enzyme activity below -100 degrees C. At even lower temperatures, enzyme activity may be limited by the effect of solvent fluidity on substrate/product diffusion, but overall there is no evidence that any intrinsic enzyme property imposes a lower temperature limit for enzyme activity.     

Removal of Linear and Branched Alkylphenols from Aqueous Solutions with Horseradish Peroxidase

Alkylphenols were effectively treated with horseradish peroxidase at pH 7.0 and 30 °C in the presence of H₂O₂ and poly(ethylene glycol) irrespective of the relative position or isomeric form of the alkyl chains. Water-insoluble oligomer precipitates were readily filtered out after enzymatic treatment, and transparent and colorless solutions were obtained for all p- and m-alkylphenols used.     

The role of temperature in enhancing the insecticidal activity of Bacillus thuringiensis against Spodoptera littoralis larvae

The present study scrutinised how far temperature would affect the velocity of the insecticidal activity of Bacillus thuringiensis, as the rapidity of pest control achievements is of a great concern. Third instar Spodoptera littoralis larvae were treated with Bt at three concentration levels under five different temperatures (15°C, 20°C, 25°C, 30°C and 35°C). LT₅₀s were evaluated in each case. The LT₅₀ values showed various levels of reductions as temperature and/or Bt concentration increased, indicating that the velocity of mortality (1/LT₅₀) and/or the rapidity of Bt activity was almost temperature dependant. However, relatively high and low reduction percentages in the LT₅₀ values on the elevation of 5°C were obtained at lower and higher temperature ranges, respectively. The temperature coefficient, Q ₁₀ values, determined within narrow ranges (5°C) showed great reductions when temperature increased from 15°C to 20°C at all Bt concentrations. Raising temperature by 5°C above 20°C or 25°C almost caused similar Q ₁₀ values indicating constant increase in the response of Bt activity within 20–30°C temperature range. Q ₁₀ values over 30°C were comparatively very low. This proved that decrease in Q ₁₀ values due to the rise of temperature was dependant on the starting temperature.     

The Effect of Temperature on the Bioventing of Soil Contaminated with Toluene and Decane

The effect of temperature on evaporation and biodegradation rates during soil bioventing (SBV) was studied for a mixture of toluene and decane in bench-scale soil columns at a continuous air flow and consecutively at two different flow rates. The effect of temperature on SBV was monitored by GC headspace analysis of contaminant, CO₂ and O₂ concentrations in the soil gas over time. Separation of evaporation and biodegradation processes into three different phases based on their rates was used together with Q₁₀ and E₁₀ (values that give the factor by which biodegradation and evaporation rates increase when the temperature is raised by 10 degrees) to compare quantitatively the removal kinetics at 10 and 20°C. Adsorption of toluene and decane onto soil (a phase partitioning process) at 20 and 10°C was described with linear Freundlich isotherms. A temperature decrease from 20 to 10°C resulted in an increase of soil-air partitioning coefficients by a factor of 1.8 and of 2.1 for toluene and decane, respectively. The mean Q₁₀ value for the biodegradation of toluene was found to be 2.2 for a temperature rise from 10 to 20°C. A toluene content in the soil gas above 75% of the saturation concentration inhibited biodegradation at both temperatures. The SBV efficiency was dependent on temperature with respect to remediation time. SBV at 20°C resulted in a 99.8% and a 98.7% reduction of toluene and decane initial concentrations, respectively. To reach similar results at 10°C, about 1.6 times as much time and 1.4 times as much air were required; however, at both temperatures the total amounts of biodegraded hydrocarbons were approximately the same. The evaporation-to-biodegradation ratios at 20°C were 82.5:17.5 for toluene and 16:84 for decane, whereas at 10 °C they were 71:29 and 2:98, respectively. A comparison of Q₁₀ values showed that, except during the initial phase of SBV, only a modest decrease in biodegradation rates should be expected after a decrease in temperature from 20 to 10°C. Flow rate reduction had a significant impact on the toluene evaporation rate at a higher temperature, whereas for decane this rate was only slightly affected by temperature. In contrast to decane, the ratio between toluene vapor pressures at 20 and 10°C may be used to predict the removal of toluene by evaporation during the above-mentioned phases of SBV, when evaporation is important.     

Catalytic and kinetic properties of purified high-affinity cyclic AMP phosphodiesterase from dog kidney

High-affinity cyclic AMP phosphodiesterase purified to homogeneity from dog kidney was studied with respect to its stability, its catalytic and kinetic properties, and its sensitivity to pharmacological agents. The enzyme was shown to rapidly lose activity upon dilution to low protein concentrations in aqueous media, but this activity loss was largely prevented by the presence of bovine serum albumin or ethylene glycol. Similarly, maximum activity required bovine serum albumin to be present during incubation for activity analysis. Enzyme activity required a divalent cation; Mg²⁺, Mn²⁺, and Co²⁺ each supported activity, but highest activity was obtained with Mg². The temperature optimum ranged from 30 to 45 °C and depended on substrate concentration; the E_a = 10,600 cal/mol. The pH optimum of the enzyme was broad, with a maximum from pH 8.0 to 9.5. The enzyme exhibits linear Michaelis-Menton kinetics for hydrolysis of cyclic AMP at all substrate concentrations tested and for hydrolysis of cyclic GMP at > 20 μm. The K_m for cyclic AMP hydrolysis was 2 μm, and that for cyclic GMP hydrolysis was 312 μm. The K_i values for the competitive inhibition of hydrolysis of each substrate by the other were similar to their K_m values suggesting a single active site. Cyclic AMP hydrolysis was weakly inhibited by cyclic GMP, cyclic IMP, adenine, and adenosine, but was not inhibited by the mono-, di, or trinucleotides of adenosine, guanosine, or inosine. Activity was competitively inhibited with K_i values in the micromolar range by drugs representative of methylxanthines, isoquinolines, pyrazolopyridines, imidazolidinones, triazolopyrimidines, pyridylethylenediamines, phenothiazines, and calcium antagonists. The results are discussed with reference to the similarities and differences between high- and low-affinity phosphodiesterase forms.     

The temperature-dependent self-association of beta-lactoglobulin C in glycine buffers

The self-association of β-lactoglobulin C at low pH (ca. 2.5) in glycine buffers has been studied at four temperatures, 10, 16, 20, and 25 °C, by low- and high-speed sedimentation equilibrium experiments. One buffer had an ionic strength of 0.1 and the other an ionic strength of 0.2. With either buffer the concentration dependence of the apparent weight average molecular weight, M_wa, was characteristic of a nonideal self-association. Like its genetic variants, β-lactoglobulin A and B, the self-association of β-lactoglobulin C increased with decreasing temperature; however, at the same temperature the association was always stronger in the buffer having the higher ionic strength. Several models were used to test the self-association, and a monomer-dimer self-association seemed to describe the self-association best with either buffer. Values of the association equilibrium constant, K₂, and the second virial coefficient, BM₁, are reported at each temperature for both series of experiments. Values of the thermodynamic functions, ΔG °, ΔH °, and ΔS °, are also reported for these experiments.     

Membrane energization at subzero temperatures: calcium uptake and oxonol-V responses

The use of aprotic solvents for preserving the electron transport properties of mitochondria at subzero temperatures is based upon the use of binary water and ethylene glycol mixtures or upon ternary and quaternary mixtures that include dimethyl sulfoxide and the lower aliphatic alcohols. In order to better preserve the respiratory control properties of mitochondria at subzero temperatures, detailed studies have been made of the effects of these mixtures on the respiratory control and electron transport from NADH or succinate of mitochondrial preparations. It is found that ADP is not metabolized at a measurable rate below 0 °C, but that Ca²⁺ is rapidly taken up and can thus be used to assay respiratory control ratios down to ?8 °C. In the region below ?8 °C the charge-sensitive probe oxonol-V has been used to evaluate energy coupling. By using Ca²⁺ to stimulate respiration at 0 °C good results are obtained with ethylene glycol/water alone and optimal results are obtained with a quaternary mixture. A mixture that freezes at ?21 °C gives about 50% inhibition of the respiratory control ratio for electron transport at 0 °C with NADH or succinate as substrates. The mixtures permit low-temperature studies of mitochondrial functions under conditions of minimal respiratory rate, including the kinetics of electron transfer reactions, the formation of intermediate compounds, and the rapid freeze-trapping of mitochondrial reactions for analytical chemistry or ³¹P NMR.     

Photosynthesis in cold acclimated leaves of plants with various degrees of freezing tolerance

The objective of this study was to compare the photosynthetic changes during cold acclimation in various plant types able to acquire different degrees of freezing tolerance. Four herbaceous and six woody plants were hardened under natural or artificial conditions and – after determination of their frost resistance (LT₅₀) – the net photosynthetic rate at an ambient CO₂ of 33 Pa (P_n33), the dependencies of P_n to light and to CO₂ and the room temperature chlorophyll a fluorescence were recorded under optimal conditions. Herbaceous plants acquired freezing tolerances to temperatures between ?10 and ?15°C when hardened at temperatures around 0°C. Most leaves fully developed prior to frost hardening exhibited typical symptoms of senescence after frost hardening. In non-senescing leaves P_n33 was reduced by 15 to 50% mainly due to a reduced stomatal conductance. After hardening at temperatures around ?10°C Brassica survived down to ?24°C, but P_n33 was almost abolished as a result of disturbances in the chloroplasts. After transferring the plants to 20/15°C P_n33 recovered completely within a few days. Woody plants hardened at temperatures around 0°C tolerated – 15 to ?36°C: P_n33 was reduced by 25 to 60% and hardly recovered at 20/15°C. Hardening at ?10°C induced a tolerance of ?32 to n33 was almost totally blocked, but at 20/15°C it returned to the values of the plants hardened at 0°C within a few days. In woody plants disturbances were invariably localized in the chloroplasts. Thus, conifers, and especially Pinus cembra, can survive much lower temperatures than herbaceous plants and, at the same level of freezing tolerance, exhibit appreciably less restriction in relative P_n33.