Relationship between Phospholipid Breakdown and Freezing Injury in a Cell Wall-Less Mutant of Chlamydomonas reinhardii

The effects of freezing and thawing on a cell wall-less mutant (CW15+) of Chlamydomonas reinhardii were investigated by monitoring enzyme release, cell viability, cell ultrastructure, and lipid composition. Cells suspended in Euglena gracilis medium were extremely susceptible to freezing injury, the median lethal temperature in the presence of extracellular ice being −5.3°C. Cell damage was associated with a release of intracellular enzymes and massive breakdown of cellular organization. Changes in phospholipid fatty acid composition consistent with either a peroxidation process or phospholipase A₂ activity were evident, but the time course of these changes showed clearly that alterations in phospholipid fatty acid composition were a secondary, pathological event and not the the primary cause of freeze-thaw injury in Chlamydomonas reinhardii CW15+.     

Aliphatic compounds in some lichens

The presence of free fatty acids in Cetraria nivalis and Cladonia gonecha has been demonstrated. Saturated wax esters occur in Cetraria cucullata and C. nivalis, whilst free alkanols were found in C. cucullata. Triglycerides in C. nivalis were detected by their MS fragmentation patterns. Hexa(α-hydroxyisovalerate) is present in C. nivalis and Cl. gonecha, and the presence of ribitol in C. nivalis was demonstrated by identification of its acetonide in an acetone extract.     

The Effects of Temperature on 86Rb Uptake by Two Species of Chlamydomonas (Chlorophyta, Chlorophyceae)

⁸⁶Rb uptake was examined in two species of unicellular greenalgae, Chlamydomonas nivalis isolated from snow, and a cellwall-less mutant of the temperate freshwater Chlamydomonas reinhardii.In C. reinhardii cells grown at 20°C and cooled rapidlyto 0°C, ⁸⁶Rb uptake was abolished. Cells cooled rapidlyto –5°C in the absence of ice accumulated ⁸⁶Rb veryrapidly but the time course of this uptake suggested non-selectiveaccumulation through a damaged plasmalemma. Cells grown at 8°Cwere viable, able to divide and motile; they showed no signsof cold-shock and ⁸⁶Rb uptake, albeit slow, was measurable at–5°C in the absence of extracellular ice. Cells ofC. nivalis grown at 20°C were damaged at sub-zero temperaturesalthough they did show an enhanced ⁸⁶Rb uptake at 0°C. Cellsgrown at 5°C were able to accumulate ⁸⁶Rb from media undercooledto -5°C in the absence of extracellular ice, and again showedenhanced uptake at 0°C. The process of acclimation to lowtemperature appears to differ in the two species. Key words: Chlamydomonas, temperature, ⁸⁶Rb uptake, membrane     

Effects of Various Rates of Freezing on the Metabolism of a Drought-tolerant Plant, the Moss Tortula ruralis

The response of the drought-tolerant moss Tortula ruralis ([Hedw.] Gaertn., Meyer, Scherb.) to freezing and thawing at controlled rates has been studied. Slow freezing (at 3 C per hour to −30 C) of hydrated T. ruralis leads to only temporary, reversible changes in metabolism. These changes can be considered to result from desiccation due to extracellular ice formation. In contrast, rapid freezing in liquid N₂ and thawing in 20 C water leads to deterioration in all aspects of metabolism studied: ribosome, protein, and ATP levels decrease, and in vivo and in vitro protein synthetic activity is lost rapidly. Such changes probably result from intracellular ice formation. Following freezing and thawing at an intermediate rate (60 C per hour), only ATP levels and in vivo protein synthesis are reduced. The protein-synthesizing apparatus (the polyribosomes) remains intact and active in an in vitro protein-synthesizing system even 24 hours after one 60 C per hour freeze-thaw cycle. These metabolic responses are discussed in terms of the two-factor hypothesis of Mazur et al. (1972 Exp. Cell Res. 71: 345-355).     

Immune reactivity of frozen-thawed murine spleen cells.

Spleen cells from mice immunized with SRBC were subjected to controlled rate freezing to ?100 °C. Complete recovery of PFC was obtained with DMSO used as the cryopreservative. Simple dilution of spleen cells in DMSO, or a single cycle of freezing and thawing in DMSO prior to short-term culture, resulted in early loss of recoverable cells. A single cycle of freezing and thawing inhibited the in vitro immune response to SRBC while having little effect on the response to TNP-T4. The in vitro blastogenic responses to LPS and PHA-P were severely reduced in cultures of frozen and thawed cells.     

Survival of tissue culture cells frozen by a two-step procedure to -196 degrees C. I. Holding temperature and time.

A two-step freezing procedure has been examined in order to separate some of the causes of damage following freezing and thawing. Different holding temperatures and times have been studied during the freezing of Chinese hamster tissue culture cells in dimethyl sulphoxide (5%, $\text{v}\text{v}$). Damage following rapid cooling to, time at, and thawing from different holding temperatures was found to increase at lower holding temperatures and at longer times. Damage on subsequent cooling from the holding temperature to ?196 °C and thawing was found to diminish at lower holding temperatures and longer times. The net result was that optimal survival from ?196 °C was obtained after 10 min at ?25 °C. Protection against the second step of cooling to ?196 °C was acquired at the holding temperature itself and was absent at ?15 °C without freezing.It seems that this technique will allow the different phases of freezing injury to be separated. These phases may include thermal shock to the holding temperature, hypertonic damage at the holding temperature and dilution shock on thawing from ?196 °C.     

Influence of concanavalin A on autolysis of gametes from Chlamydomonas reinhardii

The phytohemagglutinin concanavalin A inhibited zygote formation of Chlamydomonas reinhardii. 15–50 μg lectin/ml not only interfered with the mating reaction, but also with cell wall lysis of gametes and zoospores in a crude autolysin preparation gained from copulating gametes. Further, the structure of cell walls shed into the medium after autolysis in the course of the mating reaction and after lysis “from without” in the crude autolysin preparation was stabilized by Con A. Therefore, it must be assumed that the lectin inhibited zygote formation of C. reinhardii by interfering with autolysis of the cell walls of the gametes. Though Con A inhibited the lytic processes of C. reinhardii, an activation of the autolytic system in ⊖ gametes by the lectin was found to compete with its inhibitory reaction. Con A induced autolysis of ⊖ gametes was dependent on adherence of the cells by their flagella to the surface of the culture vessel or the liquid medium and did not occur in cultures stirred by rotation. The interferences of Con A with the autolytic system of C. reinhardii were inhibited by methyl-α-d-mannopyranoside and to a lesser degree by glucose, indicating that the carbohydrate binding sites of the lectin were involved in its reactions with the cells.     

Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques

The factors that affect the survival of mouse lymphocytes throughout a procedure for storage at ?196 °C have been studied both for the improvement of recovery and the possible extension to the mouse system of cell selection by freezing. After thawing, the survival of cells cooled at different rates in dimethyl sulphoxide (DMSO, 5 or 10%, $\text{v}\text{v}$) was assessed from the [³H]thymidine incorporation in response to phytohaemagglutinin and concanavalin A. Before freezing the protection against freezing damage increased with time (up to 20 min) in DMSO (5%, $\text{v}\text{v}$) at 0 °C. Superimposed upon this effect was toxicity due to the DMSO. During freezing and thawing the cooling rate giving optimal survival was 8 to 15 °C/min for cells in DMSO (5%) and 1 to 3 °C/min for DMSO (10%). Omission of foetal calf serum was detrimental. Rapid thawing (>2.5 °C/min) was superior to slow thawing. After thawing dilution at 25 or 37 °C greatly improved cell survival compared with 0 °C; at 25 °C survival was optimal (75%) at a moderate dilution rate of 2.5 min for a 10-fold dilution in FCS (10%, $\text{v}\text{v}$) followed by gentle centrifugation (50g).Dilution damage during both thawing and post-thaw dilution may be due to osmotic swelling as DMSO and normally excluded solutes leave the cell. The susceptibility of the cell membrane to dilution damage may also be increased during freezing. The need to thaw rapidly and dilute at 25 °C after thawing is probably due to a decrease in dilution stress at higher temperatures. Optimisation of dilution procedures both maximised recovery and also widened the range of cooling rates over which the cells were recovered. These conditions increase the possibility of obtaining good recovery of a mixed cell population using a single cooling procedure. Alternatively, if cell types have different optimal cooling rates, stressful dilution may allow their selection from mixed cell populations.     

Effects of freezing to −196 °C and thawing on Setaria lutescens seeds

The effects of single and repeated freezing and thawing of Setaria lutescens seeds in liquid nitrogen were investigated. One freeze to ?196 °C followed by a slow thaw, increased seed germination from 40 to 70%, but additional freeze-thaw cycles reduced germination to 30%. Using a scanning electron microscope, evidence was produced that seed coat cracking did not cause either initial increased, or subsequent reduced germination. Observations with a transmission electron microscope revealed that disruption of the integrity of lipid bodies accompanied increased damage from repeated freezing at ?196 °C and thawing. Repeated freezing and thawing of seeds stored in liquid nitrogen should be done with care to avoid loss of the germplasm.     

Herbicide-binding to thylakoid membranes of a DCMU-resistant mutant of Chlamydomonas reinhardii

The specific binding of DCMU and atrazine to the thylakoid membranes of a uniparentally inherited DCMU-resistant mutant dr-416 of Chlamydomonas reinhardii was measured. Whole cells of the mutant can tolerate a 15-fold concentration of DCMU as compared to the parent strain. The same tolerance is found for the photosystem II activity of isolated thylakoid membranes. The mutant is not resistant against atrazine. In equilibrium-binding studies with [¹⁴C]atrazine and unlabelled DCMU, the specific binding for atrazine was found to be identical in the mutant and in the parent strain. The competitive binding of DCMU is significantly weaker for membranes of the mutant than of the parent strain, the equilibrium dissociation constants being 2.0 × 10^?7 M and 3.8 × 10^?8 M, respectively.     

Biochemical characterization of a singular mutant of nitrate reductase from Chlamydomonas reinhardii. New evidence for a heteropolymeric enzyme structure

A singular mutant strain from Chlamydomohas reinhardii defective in nitrate reductase has been characterized. Mutant 301 possesses an ammonia-repressible NAD(P)H-cytochrome c reductase with the same charge and size properties as the low molecular weight ammonia-repressible diaphorase present in the wild-type strain 6145c and is also able to reconstitute NAD(P)H-nitrate reductase activity by in vitro complementation with reduced benzyl viologen-nitrate reductase from mutant 305. Furthermore, a heat-labile costitutive molybdenum cofactor which is fuctionally active is also present in mutant 301. Mutant 301 has the two requirements exhibited by the active nitrate reductase complex from fungi, namely, NAD(P)H-cytochrome c reductase activity and molybdenum cofactor, but lacks NAD(P)H-nitrate reductase activity. This fact together with biochemical data presented from other C. reinhardii mutants strongly suggest a heteropolymeric model for the nitrate reductase complex of the alga.     

Trehalose as cryoprotectant for the freeze preservation of carrot and tobacco cells

Suspension cultures of carrot (Daucus carota, line C1), tobacco (Nicotiana tabacum, line TX1), and Nicotiana plumbaginifolia (line NP) were frozen under controlled conditions with trehalose as the sole cryoprotectant. Maximal post-thaw viability (71-74%), measured by phenosafranin dye exclusion, was obtained with the C1 cells following a 24hour pretreatment with 5 or 10% trehalose and with 40% trehalose as the cryoprotectant during freezing. TX1 cells pretreated for 24 hours with 10% trehalose and cryoprotected with 40% trehalose during freezing showed 47% viability following thawing as determined by phenosafranin dye exclusion. The NP cells required a 3 to 6 day pretreatment with 10% trehalose and 40% trehalose as a cryoprotectant at the time of freezing for the recovery of viable cells. Growing cells were recovered when the C1 and NP cells treated as described were plated on agar-solidified medium following thawing.     

Estudios de la viabilidad y la vitalidad frente al congelado de la levadura probiótica Saccharomyces boulardii: efecto del preacondicionamiento fisiológico

The aim of this study was to evaluate the vitality and viability of the probiotic yeast Saccharomyces boulardii after freezing/thawing and the physiological preconditioning effect on these properties. The results indicate that the specific growth rate (0.3/h^?1) and biomass (2-3 × 10⁸ cells/ml) of S. boulardii obtained in flasks shaken at 28 °C and at 37 °C were similar. Batch cultures of the yeast in bioreactors using glucose or sugar-cane molasses as carbon sources, reached yields of 0.28 g biomass/g sugar consumed, after 10 h incubation at 28 °C; the same results were obtained in fed batch fermentations. On the other hand, in batch cultures, the vitality of cells recovered during the exponential growth phase was greater than the vitality of cells from the stationary phase of growth. Vitality of cells from fed-batch fermentations was similar to that of stationary growing cells from batch fermentations. Survival to freezing at –20 °C and subsequent thawing of cells from batch cultures was 0.31% for cells in exponential phase of growth and 11.5% for cells in stationary phase. Pre-treatment of this yeast in media with water activity (a_w) 0.98 increased the survival to freezing of S. boulardii cells stored at –20 °C for 2 months by 10 fold. Exposure of the yeast to media of reduced a_w and/or freezing/thawing process negatively affected cell vitality. It was concluded that stress conditions studied herein decrease vitality of S. boulardii. Besides, the yeast strain studied presented good tolerance to bile salts even at low pH values.     

Permeability changes in membranes of Neurospora crassa after freezing and thawing.

Conidia of Neurospora crassa which are in different physiological states show different rates of survival after freezing and thawing. [¹⁴C]adenine uptake by frozen and thawed conidia in different physiological states show a correlation with their survival. The uptake method was extended to study the survival of mycelium in log phase and stationary phase. From the uptake data it appears that log phase mycelium is extremely sensitive to all rates of freezing and thawing studied, while the stationary phase mycelium showed slight tolerance to freezing, if freezing was done at a slow rate. A study of the efflux of labeled compounds from the conidia in various physiological states or from the mycelia after freezing and thawing showed that, although efflux followed the same general trend as survival in conidia, it did not relate to the survival in mycelium, suggesting that the death of conidia or mycelium in the freeze-thaw treatments is not due to efflux of compounds.     

Role of Growth Temperature in Freeze-Thaw Tolerance of Listeria spp.

The food-borne pathogen Listeria monocytogenes can grow in a wide range of temperatures, and several key virulence determinants of the organism are expressed at 37°C but are strongly repressed below 30°C. However, the impact of growth temperature on the ability of the bacteria to tolerate environmental stresses remains poorly understood. In other microorganisms, cold acclimation resulted in enhanced tolerance against freezing and thawing (cryotolerance). In this study, we investigated the impact of growth temperature (4, 25, and 37°C) on the cryotolerance of 14 strains of L. monocytogenes from outbreaks and from food processing plant environments and four strains of nonpathogenic Listeria spp. (L. welshimeri and L. innocua). After growth at different temperatures, cells were frozen at −20°C, and repeated freeze-thaw cycles were applied every 24 h. Pronounced cryotolerance was exhibited by cells grown at 37°C, with a <1-log decrease after 18 cycles of freezing and thawing. In contrast, freeze-thaw tolerance was significantly reduced (P < 0.05) when bacteria were grown at either 4 or 25°C, with log decreases after 18 freeze-thaw cycles ranging from 2 to >4, depending on the strain. These findings suggest that growth at 37°C, a temperature required for expression of virulence determinants of L. monocytogenes, is also required for protection against freeze-thaw stress. The negative impact of growth at low temperature on freeze-thaw stress was unexpected and has not been reported before with this or other psychrotrophic microorganisms.Listeria monocytogenes remains a leading cause of deaths due to food-borne illness in the United States and other industrialized nations. Neonates, pregnant women, and immunocompromised people are at high risk for infection (15, 28, 37). Outbreaks of listeriosis tend to involve a relatively small number of closely related strains (“epidemic clones”), primarily of serotype 4b. Several major outbreaks have been attributed to epidemic clone I (ECI) and epidemic clone II (ECII), both of serotype 4b (5, 21).Unlike most other human food-borne pathogens, L. monocytogenes grows over a wide temperature range (1 to 45°C), with optimal growth at approximately 37°C (33). It has been known for some time that expression of several key virulence genes, including hly, encoding the hemolysin listeriolysin O and actA, encoding a protein that mediates actin polymerization required for intracellular pathogenesis, is optimal at 37°C but severely repressed at temperatures below 30°C (22). In contrast, flagellin, motility, and chemotaxis genes are repressed at 37°C but optimally expressed at temperatures below 25°C (9, 10).For an organism such as L. monocytogenes, which is commonly found in the environment but can also colonize and infect warm-blooded animals, temperature is likely to serve as a major signal differentiating environmental from vertebrate host-associated habitats (19). However, with the exception of the thermoregulated phenotypes described above (virulence factor production, motility, and chemotaxis), the impact of growth at different temperatures on specific responses and adaptations of the pathogen remains poorly understood.L. monocytogenes may be exposed to freezing as well as thawing in the course of its existence in natural environments (e.g., soils and water in temperate or cold regions), as well as during the storage and preservation of foods. The organism has been repeatedly isolated from frozen foods (e.g., ice cream) (7). After freezing (−18°C) in laboratory media or in foods and a single thawing cycle, survival depended on strain, freezing medium, and the presence of glycerol as cryoprotectant (13, 14). The freezing and thawing of bacteria grown at 30°C, in combination with essential oil, has been explored as one means to reduce the pathogen in foods (8). The possible role of the general stress sigma factor (sigma B) in survival of bacteria grown at 30°C and exposed to repeated freezing and thawing was also investigated (43). However, there is a surprising dearth of information on the possible impact of growth temperature on tolerance of L. monocytogenes to repeated freezing and thawing (cryotolerance).Studies with another gram-positive psychrotrophic bacterium (Exiguobacterium sibiricum and other Exiguobacterium spp.) revealed that growth in the cold (4°C), or growth on solid media regardless of temperature, resulted in increased tolerance of the bacteria against repeated freezing and thawing (40). It is not known whether low temperature or surface-associated growth may exert similar impacts on the cryotolerance of L. monocytogenes. The objective of the present study was to investigate the impact of growth temperature (4, 25, and 37°C) and of planktonic versus agar growth of L. monocytogenes on protection of the bacteria against repeated freezing and thawing.     

Phospholipid degradation in frozen plant cells associated with freezing injury

A striking degradation of phosphatidylcholine into phosphatidic acid was observed in the cortical tissues of less hardy poplar (Poplus euramericani cv. gelrica), when the tissues were frozen below a lethal temperature. No change in phospholipids was detected during freezing or even after thawing in the cortical tissues of hardy poplar which survived slow freezing to −30 C or even immersion in liquid N₂ after prefreezing to −50 C. The degradation of phosphatidylcholine during freezing appears to be intimately associated with freezing injury.     

Cryopreservation of islets of Langerhans

Development of techniques for cryopreservation of pancreatic islets of Langerhans could potentially allow for increased freedom from the time restrictions presently affecting viability in islet cell transplantation. While several investigators have attempted islet cell freezing and have obtained favorable in vitro results after thawing, there have been few reported in vivo successes with islets transplanted after freezing. We have developed a simple system for freezing islet cell pancreatic fragments to ?196 °C and have either stored them in liquid nitrogen for 24 hr or immediately thawed the islets prior to transplantation. In addition, antilymphoblast globulin has been used as graft pretreatment modality in order to modify islet cell immunogenicity. We found that ALG was effective in prolongation of graft survival after freezing as well as on fresh nonfrozen transplants. The use of freezing and ALG appears, therefore, to have a favorable effect on the immunogenicity of the pancreatic islet cell allograft.     

Xylem pressure potential and chlorophyll fluorescence as indicators of freezing survival in black locust and Western hemlock seedlings

Xylem pressure potential was determined using the Scholander pressure chamber on stems of cold hardened and non-hardened black locust (Robinia pseudoacacia L.) seedlings following freezing to various nonlethal and lethal temperatures and subsequent thawing. Correlation was found between immediate xylem pressure potential and long-term seedling survival. Chlorophyll fluorescence transients were monitored using needles of western hemlock (Tsuga heterophylla (Raf.) Sarg.) seedlings following freezing to various non-lethal and lethal temperatures and subsequent thawing. Immediate and repeatable differences in fluorescence transients correlated with long-term seedling survival. Methodology is described and correlations discussed relative to using either chlorophyll fluorescence or xylem pressure potential as an immediate indicator of long-term freezing survival in woody plant seedlings.     

Reduced Inorganic Carbon Transport in a CO(2)-Requiring Mutant of Chlamydomonas reinhardii

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated that is deficient in inorganic carbon transport. This mutant strain, designated pmp-1-16-5K (gene locus pmp-1), was selected on the basis of a requirement of elevated CO₂ concentration for photoautrophic growth. Inorganic carbon accumulation in the mutant was considerably reduced in comparison to wild type, and the CO₂ response of photosynthesis indicated a reduced affinity for CO₂ in the mutant. At air levels of CO₂ (0.03-0.04%), O₂ inhibited photosynthesis and stimulated the synthesis of photorespiratory intermediates in the mutant but not in wild type. Neither strain was significantly affected by O₂ at saturating CO₂ concentration. Thus, the primary consequence of inorganic carbon transport deficiency in the mutant was a much lower internal CO₂ concentration compared to wild type. From these observations, we conclude that enzyme-mediated transport of inorganic carbon is an essential component of the CO₂ concentrating system in C. reinhardii photosynthesis.     

Photoinhibition in the Antarctic moss Grimmia antarctici Card when exposed to cycles of freezing and thawing

Freezing and thawing of the endemic moss species Grimmia antarctici Card, caused photoinhibition. When snow cover was removed from moss in the field, resulting in exposure to fluctuating temperatures and light conditions, photoinhibition, measured as a reduction in the ratio of variable to maximum chlorophyll a fluorescence (F_v/F_m), was observed. The extent of photoinhibition was highly variable and appeared to be reversible during periods of warmer temperatures. A series of controlled laboratory studies found that the light conditions that prevail between freezing and thawing events influenced the recovery from photoinhibition observed during freezing and thawing, with low light conditions facilitating the greatest rates of recovery. After four cycles of freezing and thawing, recovery from photoinhibition in hydrated moss was achieved within 12 h of transfer to 5°C and 15 μmol quanta m^?2 s^?1. These results favour the hypothesis that photoinhibition observed during freezing represents a protective process involving the down-regulation of photo-system II when photosynthetic carbon assimilation is limited by low temperatures.