Chilling-Induced Lipid Degradation in Cucumber (Cucumis sativa L. cv Hybrid C) Fruit

Chilling at 4°C in the dark induced lipid degradation in cucumber (Cucumis sativa L.) fruit upon rewarming at 14°C. Rates of ethane evolution by fruits rewarmed after 3 days of chilling were up to four-fold higher than those evolved by unchilled (14°C) fruits (0.02-0.05 picomoles gram fresh weight⁻¹ hour⁻¹). This potentiation of lipid peroxidation occurred prior to irreversible injury (requiring 3 to 7 days of chilling) as indicated by increases in ethylene evolution and visual observations. Decreases in unsaturation of peel tissue glycolipids were observed in fruits rewarmed after 3 days of chilling, indicating the plastids to be the site of the early phases of chilling-induced peroxidation. Losses in unsaturation of tissue phospholipids were first observed only after chilling for 7 days. Phospholipase D activity appeared to be potentiated in fruits rewarmed after 7 days of chilling as indicated by a decrease in phosphatidylcholine (and secondarily phosphatidylethanolamine) with a corresponding increase in phosphatidic acid. These results indicate that lipid peroxidation may have a role in conferring chilling injury.     

Survival of Plant Tissue at Super-Low Temperatures. IV. Cell Survival with Rapid Cooling and Rewarming

Thin unmounted cortical tissue sections from winter twigs of the mulberry tree were held with a thin forceps and rapidly immersed in liquid nitrogen from room temperatures without prefreezing. They were rewarmed; rapidly in water at 10° to 40°, or slowly, in air at room temperatures. In those sections rapidly rewarmed, all survived. None survived in those sections rewarmed slowly in air.

Tissue sections mounted between coverglasses with water were extracellulary prefrozen at the temperatures low enough to dehydrate almost all of the freezable water in cells. These sufficiently prefrozen cells could survive immersion in liquid nitrogen, and the survival value was very little affected by the rates of cooling to and rewarming from super-low temperatures. With insufficient prefreezing at higher temperatures, however, the rewarming process seriously influenced the survival value of cells frozen at super-low temperatures. Slow rewarming in air destroyed all of the cells, while rapid rewarming in water at 30° did not affect them. An abrupt decrease in the survival value in insufficiently prefrozen cells during rewarming was also observed at temperatures above approximately −50° following immersion in liquid nitrogen. Very little decrease in the survival value was observed in any of the cells that had been sufficiently prefrozen.

These results indicate that cells which are insufficiently prefrozen may contain freezable water which nucleates during rapid cooling in liquid nitrogen and then grows during the subsequent slow rewarming into ice masses which destroy the viability of the cells. Such fatal intracellular freezing rarely occurs in sufficiently prefrozen cells, irrespective of the rate of cooling to or rewarming from super-low temperatures.

     

Survival of Plant Tissue at Super-Low Temperature VI. Effects of Cooling and Rewarming Rates on Survival

The survival rates of the cortical parenchymal cells of mulberry tree were determined as a function of cooling and rewarming rates. When cooling was carried out slowly at 1° to 15° per minute, all of the cells still remained viable even when rewarmed either rapidly or slowly. Survival rates gradually decreased to zero as the cooling rate increased from about 15° to 2000° per minute. In the intermediate cooling rates, when the cells were cooled at the rates lower than 14° per minute, from −2.2° to about −10°, these cells could survive subsequent rapid cooling and rewarming.

However, at cooling rates above 1000° per minute and with rapid rewarming, the effect of cooling rate reversed and survival increased, reaching a maximum at about 200,000° per minute. As the cooling rate increased above 15° per minute, survival rates became increasingly dependent on the rewarming rate, with rapid rewarming becoming less deleterious than slow rewarming.

The temperature range at which damage occurred during rewarming following removal from liquid nitrogen and in which growth rate of ice crystallization was greatest, was −30° to −40°. The survival rates even in the prefrozen cells at −30° decreased considerably by keeping them at −30° for 10 minutes after removal from liquid nitrogen. This fact indicates that intracellular freezable water remains to some degree even in the prefrozen cells at −30°. After removal from liquid nitrogen, all cells retained their viability, when they were passed rapidly through a temperature range between −50° and −2.5° within about 2 seconds, namely at the rates greater than 1000° per minute.

These observations are explained in terms of the size of the crystals formed within the cortical cells.

     

Effects of Mild Cold Shock (25°C) Followed by Warming Up at 37°C on the Cellular Stress Response

Temperature variations in cells, tissues and organs may occur in a number of circumstances. We report here that reducing temperature of cells in culture to 25°C for 5 days followed by a rewarming to 37°C affects cell biology and induces a cellular stress response. Cell proliferation was almost arrested during mild hypothermia and not restored upon returning to 37°C. The expression of cold shock genes, CIRBP and RBM3, was increased at 25°C and returned to basal level upon rewarming while that of heat shock protein HSP70 was inversely regulated. An activation of pro-apoptotic pathways was evidenced by FACS analysis and increased Bax/Bcl2 and BclX_S/L ratios. Concomitant increased expression of the autophagosome-associated protein LC3II and AKT phosphorylation suggested a simultaneous activation of autophagy and pro-survival pathways. However, a large proportion of cells were dying 24 hours after rewarming. The occurrence of DNA damage was evidenced by the increased phosphorylation of p53 and H2AX, a hallmark of DNA breaks. The latter process, as well as apoptosis, was strongly reduced by the radical oxygen species (ROS) scavenger, N-acetylcysteine, indicating a causal relationship between ROS, DNA damage and cell death during mild cold shock and rewarming. These data bring new insights into the potential deleterious effects of mild hypothermia and rewarming used in various research and therapeutical fields.     

Safety Precautions to be Observed with Cooled Premixed Gases

The gas delivered from a cylinder of Entonox (50% nitrous oxide and 50% oxygen) may not contain adequate quantities of oxygen if the cylinder has been cooled and then allowed to warm up in a vertical position.The composition of gas delivered from cylinders cooled to not lower than –40° C. remains unaltered if the cylinders are rewarmed for 24 hours in a horizontal position at a temperature of not less than 5° C.Cylinders of Entonox delivered in cold weather should be stored in a horizontal position at 5° C. or more for at least 24 hours before use.     

Fructan metabolism in wheat in alternating warm and cold temperatures

The objective of this research was to develop a system in which the direction of fructan metabolism could be controlled. Three-week-old wheat seedlings (Triticum aestivum L. cv Caldwell) grown at 25°C were transferred to cold temperature (10°C) to induce fructan synthesis and then were transferred to continuous darkness at 25°C after defoliation and fructan degradation monitored. The total fructan content increased significantly 1 day after transferring from 25°C to 10°C in both leaf blades and the remainder of the shoot tissue, 90% of which was leaf sheath tissue. Leaf sheaths contained higher concentrations of fructan and greater portions of high molecular weight fructan than did leaf blades. Fructan content in leaf sheaths declined rapidly and was gone completely within 48 hours following transfer to 25°C in darkness. In leaf blades the invertase activity fluctuated during cold treatment. The activity of sucrose:sucrose fructosyl transferase increased markedly during cold treatment, while fructan hydrolase activity decreased slightly. In leaf sheaths, however, the activity of invertase decreased rapidly upon transfer to cold temperature and remained low. Trends in sucrose:sucrose fructosyl transferase and hydrolase activity in sheaths were the same as those of leaf blades. Sheath invertase and hydrolase activity increased when plants were transferred back to darkness at 25°C, while sucrose:sucrose fructosyl transferase activity decreased. These results indicate that changing leaf sheath temperature can be utilized to control the direction of fructan metabolism and thus provide a system in which the synthesis or degradation of fructan can be examined.     

Cold Injury in Civil Disaster

Patients exposed to cold environment following a disaster may be suffering from local cold injury or from systemic cold injury (accidental hypothermia). The treatment of the former is well established and consists of rapid rewarming of the frozen parts and physical therapy; early amputation is not advisable. Recommendations for the assessment and treatment of total body cooling, based on case reports of accidental hypothermia, on the results of animal experiments and on the clinical experience with induced hypothermia, are not well established and are controversial. Since, at temperatures below 30° C., death may be caused by cardiac arrhythmias and since, at this low level, spontaneous rewarming may not be possible, active rewarming is recommended at this stage. At a higher temperature level, vigorous warming is dangerous, and the patient''s own regulatory mechanisms should be allowed to restore the temperature.     

Effect of cold acclimation on the incidence of two forms of freezing injury in protoplasts isolated from rye leaves

The freezing tolerance and incidence of two forms of freezing injury (expansion-induced lysis and loss of osmotic responsiveness) were determined for protoplasts isolated from rye leaves (Secale cereale L. cv Puma) at various times during cold acclimation. During the first 4 weeks of the cold acclimation period, the LT₅₀ (i.e. the minimum temperature at which 50% of the protoplasts survived) decreased from −5°C to −25°C. In protoplasts isolated from nonacclimated leaves (NA protoplasts), expansion-induced lysis (EIL) was the predominant form of injury at the LT₅₀. However, after only 1 week of cold acclimation, the incidence of EIL was reduced to less than 10% at any subzero temperature; and loss of osmotic responsiveness was the predominant form of injury, regardless of the freezing temperature. Fusion of either NA protoplasts or protoplasts isolated from leaves of seedlings cold acclimated for 1 week (1-week ACC protoplasts) with liposomes of dilinoleoylphosphatidylcholine also decreased the incidence of EIL to less than 10%. Fusion of protoplasts with dilinoleoylphosphatidylcholine diminished the incidence of loss of osmotic responsiveness, but only in NA protoplasts or 1-week ACC protoplasts that were frozen to temperatures over the range of -5 to -10°C. These results suggest that the cold acclimation process, which results in a quantitative increase in freezing resistance, involves several different qualitative changes in the cryobehavior of the plasma membrane.     

Short and long term effects of root and shoot chilling of ransom soybean

The immediate short term effects on some physiological processes and the long term effects on morphology and reproductive development of root- and shoot-chilled soybeans (Glycine max L. cv Ransom) were studied. Roots or shoots of 16- or 17-day-old plants were chilled at 10°C for one week, and then rewarmed to 25°C. Leaf elongation rate, net CO₂ uptake rate, and stomatal conductance decreased during root or shoot chilling. Root chilling had only temporary effects on water relations, while shoot chilling caused large changes in potentials during chilling. Most processes measured returned to control levels after two days of rewarming. Root-chilled plants harvested 90 days after emergence were similar in morphology and seed weight to controls. Shoot-chilled plants showed a large increase over controls in axillary branch growth, but an early abortion of flowers and a delayed resumption of flowering caused a 78% reduction in seed weight. Root chilling in this study was found to have little or no long term effect on the plants, while shoot chilling caused significant changes in vegetative morphology, and a delay in flowering and subsequent pod filling.     

The Effect of Temperature on the Uptake of Radiosulfate by Rat Renal Tissue from Radiosulfate-Containing Solutions in Vitro

Kidney cortex slices incubated in vitro at 0°C. accumulate radiosulfate from the incubation medium. This process differs from the previously described uptake of radiosulfate by renal tissue incubated at 38°C., for instance, in the lesser sensitivity of the uptake at 0°C. to differential effects of Na⁺ as compared with K⁺ ions, and of sucrose as compared with glucose. Phlorizin inhibits radiosulfate accumulation at 0°C., whereas it enhances the uptake at 38°C. Effects of the cations K⁺ and Na⁺ and of phlorizin at temperatures intermediate between 0° and 38°C. have been studied. Parallels have been noted between the accumulative processes for radiosulfate of kidney slices maintained at 0°C. and of mitochondria isolated from rat liver and kidney cortex. These similarities may be attributed to an important role of radiosulfate uptake by mitochondria in slice accumulation of radiosulfate in the cold.     

Influence of Cold Stress on the Preliminary Enrichment Time Needed for Detection of Enterohemorrhagic Escherichia coli in Ground Beef by PCR

The influence of cold stress at 4 and 0°C on the detection time as assessed by impedance technology (Bactometer; Biomérieux, Marcy l’Etoile, France) of different enterohemorrhagic Escherichia coli (EHEC) strains was determined. Although there is some variation in susceptibility among EHEC strains, prolonged exposure of EHEC to cold stress, i.e., 4 and 5 days at 4 and 0°C, respectively, in general significantly increased their detection time. This reflects an increase of the lag-phase time caused by cold stress. Two EHEC strains were selected to determine the minimum preliminary enrichment time that would ensure a positive PCR detection of low numbers of verotoxin-producing E. coli (VTEC; 2 to 2 × 10⁵ CFU/25 g) inoculated into ground beef (25 g) and stored at 4 or −20°C for 8 and 14 days, respectively. Incubation times of 6 and 9 h of 1 to 10 CFU/g and 1 to 10 CFU/25 g, respectively, were sufficient for PCR detection of VTEC in ground beef when analysis was performed immediately after inoculation (no cold stress). When cells are exposed to cold stress (4 or −20°C) a 24-h enrichment period is recommended. Restriction of enrichment time to 9 h under these circumstances decreases the sensitivity of PCR detection to 80 CFU/g. Hence, to obtain maximum sensitivity, PCR detection of VTEC in naturally contaminated ground beef should be performed after 24 h of enrichment.     

Effect of temperature conditioning on chilling injury of cucumber cotyledons: possible role of abscisic Acid and heat shock proteins

Endogenous abscisic acid levels and induced heat shock proteins were measured in tissue exposed for 6 hours to temperatures that reduced their subsequent chilling sensitivity. One-centimeter discs excised from fully expanded cotyledons of 11-day-old seedlings of cucumber (Cucumis sativus L., cv Poinsett 76) were exposed to 12.5 or 37°C for 6 hours followed by 4 days at 2.5 or 12.5°C. Ion leakage, a qualitative indicator of chilling injury, increased after 2 to 3 day exposure to 2.5°C, but not to 12.5°C, a nonchilling temperature. Exposure to 37°C before chilling significantly reduced the rate of ion leakage by about 60% compared to tissue exposed to 12.5°C before chilling, but slightly increased leakage compared to tissue exposed to 12.5 or 37°C and held at the nonchilling temperature of 12.5°C. There was no relationship between abscisic acid content following exposure to 12.5 or 37°C and chilling tolerance. Five heat shock proteins, with apparent molecular mass of 25, 38, 50, 70, and 80 kilodaltons, were induced by exposure to 37 or 42°C for 6 hours, and their appearance coincided with increased chilling resistance. Heat shock treatments reduced the synthesis of three proteins with apparent molecular mass of 14, 17, and 43 kilodaltons. Induction of heat shock proteins could be a possible cause of reduced chilling injury in tissue exposed to 37 or 42°C.     

Dexmedetomidine Reduces Shivering during Mild Hypothermia in Waking Subjects

Background and Purpose

Reducing body temperature can prolong tolerance to ischemic injury such as stroke or myocardial infarction, but is difficult and uncomfortable in awake patients because of shivering. We tested the efficacy and safety of the alpha-2-adrenergic agonist dexmedetomidine for suppressing shivering induced by a rapid infusion of cold intravenous fluids.

Methods

Ten subjects received a rapid intravenous infusion of two liters of cold (4°C) isotonic saline on two separate test days, and we measured their core body temperature, shivering, hemodynamics and sedation for two hours. On one test day, fluid infusion was preceded by placebo infusion. On the other test day, fluid infusion was preceded by 1.0 μg/kg bolus of dexmedetomidine over 10 minutes.

Results

All ten subjects experienced shivering on placebo days, with shivering beginning at a mean (SD) temperature of 36.6 (0.3)°C. The mean lowest temperature after placebo was 36.0 (0.3)°C (range 35.7-36.5°C). Only 3/10 subjects shivered on dexmedetomidine days, and the mean lowest temperature was 35.7 (0.4)°C (range 35.0-36.3°C). Temperature remained below 36°C for the full two hours in 6/10 subjects. After dexmedetomidine, subjects had moderate sedation and a mean 26 (13) mmHg reduction in blood pressure that resolved within 90 minutes. Heart rate declined a mean 23 (11) bpm after both placebo and dexmedetomidine. Dexmedetomidine produced no respiratory depression.

Conclusion

Dexmedetomidine decreases shivering in normal volunteers. This effect is associated with decreased systolic blood pressure and sedation, but no respiratory depression.     

Induction of Freezing Tolerance in Spinach during Cold Acclimation

Spinach (Spinacia oleracea L.) seedlings, grown in soil or on an agar medium in vitro, became cold acclimated when exposed to a constant 5°C. Plants subjected to cold acclimation, beginning 1 week postgermination, attained freezing tolerance levels similar to that achieved by seedlings that were cold acclimated beginning 3 weeks after sowing. Seedlings at 1 week of age had only cotyledonary leaves, while 3-week-old seedlings had developed true leaves. Plants grown in vitro were able to increase in freezing tolerance, but were slightly less hardy than soil-grown plants. These results suggest that spinach, a cool-season crop that begins growth in early spring when subzero temperatures are likely, can undergo cold acclimation at the earliest stages of development following germination. Axenic seedlings, grown in vitro, were used to develop a noninjurious radiolabeling technique. Leaf proteins were radiolabeled to specific activities of 10⁵ counts per minute per microgram at 25°C or 5 × 10⁴ counts per minute per microgram at 5°C over a 24 hour period. The ability to radiolabel leaf proteins of in vitro grown plants to high specific activities at low temperature, without injury or microbial contamination, will facilitate studies of cold acclimation.     

Occurrence of Shewanella algae in Danish Coastal Water and Effects of Water Temperature and Culture Conditions on Its Survival

The marine bacterium Shewanella algae, which was identified as the cause of human cases of bacteremia and ear infections in Denmark in the summers of 1994 and 1995, was detected in seawater only during the months (July, August, September, and October) when the water temperature was above 13°C. The bacterium is a typical mesophilic organism, and model experiments were conducted to elucidate the fate of the organism under cold and nutrient-limited conditions. The culturable count of S. algae decreased rapidly from 10⁷ CFU/ml to 10¹ CFU/ml in approximately 1 month when cells grown at 20 to 37°C were exposed to cold (2°C) seawater. In contrast, the culturable count of cells exposed to warmer seawater (10 to 25°C) remained constant. Allowing the bacterium a transition period in seawater at 20°C before exposure to the 2°C seawater resulted in 100% survival over a period of 1 to 2 months. The cold protection offered by this transition (starvation) probably explains the ability of the organism to persist in Danish seawater despite very low (0 to 1°C) winter water temperatures. The culturable counts of samples kept at 2°C increased to 10⁵ to 10⁷ CFU/ml at room temperature. Most probable number analysis showed this result to be due to regrowth rather than resuscitation. It was hypothesized that S. algae would survive cold exposure better if in the biofilm state; however, culturable counts from S. algae biofilms decreased as rapidly as did counts of planktonic cells.     

Radiation Resistance and Injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25°C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and −30°C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at −20°C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at −20°C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at −20°C, nor did storage at −20°C alter the cell's resistance to irradiation at 25°C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36°C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36°C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5°C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36°C for 1 day than at 5°C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

Biophysics of the inhibition of the growth of maize roots by lowered temperature

Roots of hydroponically grown maize (Zea mays cv LG11) have a greatly reduced growth rate at 5°C (0.02 millimeters per hour) compared with those at 20°C (1.2 millimeters per hour). Various physical parameters of roots growing at each temperature were compared. Turgor pressure of cells in the elongation zone increased from 0.59 ± 0.05 megapascal at 20°C to 0.82 ± 0.04 megapascal after 70 hours at 5°C; thus, growth rate was not limited by decreased pressure. On cooling, tissue plasticity (measured by Instron/tensiometer) decreased slowly over 70 hours. On rewarming to 20°C from 5°C, growth rate, turgor pressure, and tissue plasticity all returned concertedly to their original values over a period of days. However, immediately following cooling growth rate dropped rapidly from 1.8 to 0.12 millimeters per hour in 30 minutes but turgor pressure and tissue Instron plasticity remained unchanged. A plot of turgor pressure against growth rate indicated that, following cooling from 30 to 15°C, the in vivo wall extensibility of the tissue was reduced by 75%. Yield threshold was unchanged. Cells whose expansion was arrested in the long-term cold treatment do not resume growth. Root growth recovers by the expansion of cells newly produced by the meristem. Cessation of extension growth is an effect on the individual expanding cell. Growth recovery is not a reverse of this effect but requires the generation of fresh cells.     

Changes in the integrin-mediated adhesion of human neutrophils in the cold and after rewarming

Changes in the mechanical and adhesive properties of neutrophils may modify perfusion of the microcirculation in cooled tissue. We tested how integrin-mediated adhesion of isolated human neutrophils was altered by cooling, or cooling and rewarming. First, adhesion was tested in a static assay. In the presence or absence of integrin-activating agents (formyl peptide, fMLP or Mn(++)), there were significant reductions in adhesion to immobilised albumin at 10 degrees C or 0 degrees C compared to 37 degrees C, although a slight increase in adhesion was induced by fMLP or Mn(++) at 10 degrees C or 0 degrees C. If cells were cooled for 5 or 20 min at 10 degrees C and rewarmed (in the absence of activators) there was >100% increase in adhesion compared to cells held at 37 degrees C. In a flow assay, neutrophils perfused over P-selectin at 37 degrees C formed rolling attachments, but if neutrophils were cooled to 10 degrees C and rewarmed for 1 or 5 min, there was transformation to stationary adhesion, which was reversed by antibody against CD18. After 20 minutes of rewarming, rolling was restored. Cooling and rewarming did not cause de novo expression of CD11b/CD18, and so appears to transiently activate constitutively-expressed integrin. Thus, integrin-mediated adhesion may be impaired in cold tissue but on return to normal temperature, neutrophils may transiently adhere locally or in remote vessels.     

Freezing tolerance of citrus, spinach, and petunia leaf tissue : osmotic adjustment and sensitivity to freeze induced cellular dehydration

Seasonal variations in freezing tolerance, water content, water and osmotic potential, and levels of soluble sugars of leaves of field-grown Valencia orange (Citrus sinensis) trees were studied to determine the ability of citrus trees to cold acclimate under natural conditions. Controlled environmental studies of young potted citrus trees, spinach (Spinacia pleracea), and petunia (Petunia hybrids) were carried out to study the water relations during cold acclimation under less variable conditions. During the coolest weeks of the winter, leaf water content and osmotic potential of field-grown trees decreased about 20 to 25%, while soluble sugars increased by 100%. At the same time, freezing tolerance increased from lethal temperature for 50% (LT₅₀) of −2.8 to −3.8°C. In contrast, citrus leaves cold acclimated at a constant 10°C in growth chambers were freezing tolerant to about −6°C. The calculated freezing induced cellular dehydration at the LT₅₀ remained relatively constant for field-grown leaves throughout the year, but increased for leaves of plants cold acclimated at 10°C in a controlled environment. Spinach leaves cold acclimated at 5°C tolerated increased cellular dehydration compared to nonacclimated leaves. Cold acclimated petunia leaves increased in freezing tolerance by decreasing osmotic potential, but had no capacity to change cellular dehydration sensitivity. The result suggest that two cold acclimation mechanisms are involved in both citrus and spinach leaves and only one in petunia leaves. The common mechanism in all three species tested was a minor increase in tolerance (about −1°C) resulting from low temperature induced osmotic adjustment, and the second in citrus and spinach was a noncolligative mechanism that increased the cellular resistance to freeze hydration.