The kinetics of light-induced changes of C-550, cytochrome b559 and fluorescence yield in chloroplasts at low temperature

The kinetics of the photoreduction of C-550, the photooxidation of cytochrome b₅₅₉ and the fluorescence yield changes during irradiation of chloroplasts at ?196 °C were measured and compared. The photoreduction of C-550 proceeded more rapidly than the photooxidation of cytochrome b₅₅₉ and the fluorescence yield increase followed the cytochrome b₅₅₉ oxidation. These results suggest that fluorescence yield under these conditions indicates the dark reduction of the primary electron donor to Photosystem II, P680⁺, by cytochrome b₅₅₉ rather than the photoreduction of the primary electron acceptor.The photoreduction of C-550 showed little if any temperature dependence over the range of ?196 to ?100 °C. The amount of cytochrome b₅₅₉ photooxidized was sensitive to temperature decreasing from the maximal change at temperatures between ?196 to ?160 °C to no change at ?100 °C. To the extent that the reaction occurred at temperatures between ?160 and ?100 °C the rate was largely independent of temperature. The rate of the fluorescence increase was dependent on temperature over this range being 3–4 times more rapid at ?100 than at ?160 °C. At ?100 °C the light-induced fluorescence increase and the photoreduction of C-550 show similar kinetics. The temperature dependence of the fluorescence induction curve is attributed to the temperature dependence of the dark reduction of P680⁺.The intensity dependence of the photoreduction of C-550 and of the photooxidation of cytochrome b₅₅₉ are linear at low intensities (below 200 μW/cm²) but fall off at higher intensities. The failure of reciprocity in the photoreduction of C-550 at the higher intensities is not explained by the simple model proposed for the Photosystem II reaction centers.     

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.     

Absorption and circular dichroism spectra of chloroplast membrane fragments from spinach,barley and a barley mutant at room temperature and liquid nitrogen temperature

The absorption and CD spectra of chloroplast fragments from spinach, barley and a barley mutant (chlorophyll b-minus) were studied at temperatures of 23°C and ?196°C. The CD spectrum of wild type barley and spinach at ?196°C showed troughs at 640, 653, 676 and 695 nm and a maximum at 667 nm. The CD spectrum of the barley mutant at ?196°C consisted of a large trough at 684 nm, a small trough at 695 nm and a positive peak at 670 nm. A new feature observed at ?196°C but not at 23°C is the trough at 640 nm. This 640 nm CD signal is missing in the CD spectrum of the barley mutant. It is attributable to the light-harvesting chlorophyll $\text{a}\text{b}$ protein which appears to be missing in the mutant. Another new feature, the trough at 695 nm, was observed in the CD spectra of spinach, barley and the barley mutant at ?196°C. The 695 nm trough appears to be sensitive to detergents and it may be due to a labile chlorophyll a·protein complex. Possible interpretations of these data are discussed.     

Variations spectroscopiques induites par illumination et par action chimique observées à—196°C dans la chlorophylle a de Porphyridium

Spectroscopic Changes in the Chlorophyll a of Porphyridium Induced by Illumination and Chemical Action and Observed at ?196°C. Photo-oxidation of P700 by 708 nm light can take place under weak intensity (10^?6 W × cm^?2) when the medium is frozen. Spectral characteristics of “700 nm” and “690 nm” variations are accurately measured. The amplitude of the photoinduced changes of absorption are similar to those induced by chemical action. In the case of Porphyridium, an apparent increase of the extinction power of P700 at ?196°C is observed. This fact seems to be due to a diminution of the bandwith of the neighbouring pigments. Irradiation with red light (685 nm), of a relatively high intensity (10^?2 W × cm^?2), in the presence of oxygen at ?196°C, induces a slight shift (0.5 nm) of the red absorption band maximum towards longer wavelengths. This change is similar to the one promoted by ferricyanide in the dark. The origin and the functional significance of the phenomenon is discussed.     

Hybrid lethality in interspecific hybrids between Nicotiana tabacum and N. suaveolens: evidence that the Q chromosome causes hybrid lethality based on Q-chromosome-specific DNA markers

Hybrid seedlings from the cross Nicotiana tabacum × N. suaveolens express lethality at 28°C. We carried out a cross between monosomic lines of N. tabacum lacking the Q chromosome and N. suaveolens by test-tube pollination and ovule culture at 28°C. To suppress hybrid lethality, hybrid seedlings obtained were transferred to 36°C immediately after germination and cultured. We determined whether Q-chromosome-specific DNA markers were detected among hybrid seedlings. When hybrid seedlings cultured at 36°C were transferred to 28°C, hybrid seedlings in which Q-chromosome-specific DNA markers were detected expressed hybrid lethality, while hybrid seedlings in which Q-chromosome-specific DNA markers were not detected did not express hybrid lethality. From these results, we concluded that the presence of the Q chromosome of N. tabacum is related to hybrid lethality observed in crosses between N. tabacum and N. suaveolens. This is the first report that clearly demonstrates the relationship between a certain chromosome and hybrid lethality in the genus Nicotiana using chromosome-specific DNA markers. Additionally, we confirmed that the Q chromosome belongs to the S subgenome because Q-chromosome-specific DNA markers were detected only in N. sylvestris.     

Long-term stability of parameters of antioxidant status in human serum

Abstract

The antioxidant status of serum or plasma can be determined using several commercially available assays. Here, four different assays, total antioxidant status (TAS), its second-generation assay (TAS2), biological antioxidant potential (BAP), and enzymatic assay using horseradish peroxidase (EAOC), were applied on human serum samples to test the temperature stability of antioxidants, upon storage of serum for 12 months. The two or three most commonly used temperatures for storage, that is, ? 20, ? 70 (or ? 80), and ? 196°C, were selected. The general conclusion is that all assays were stable at the temperatures tested. In addition, there were almost no statistically significant differences between the samples stored at different temperatures. Only the rank order of the EAOC assay was not very good in samples stored at ? 20°C. Also three components contributing to the total antioxidant capacity, uric acid, creatinine and bilirubin, showed no statistically significant differences between the temperatures. Therefore, storage at ? 20°C is sufficient to maintain a proper assay outcome of most of the total antioxidant assays, although storage at ? 70/80°C is to be preferred for longer storage times.     

Temperature-dependent development, diapause and cold tolerance of Gratiana graminea, a potential biological control agent of Solanum viarum in Florida, USA

The objectives of this study were to examine temperature-dependent development, diapause and cold tolerance of Gratiana graminea Klug (Chrysomelidae), a candidate biological control agent of tropical soda apple, Solanum viarum Dunal (Solanaceae). Immature development was examined at six constant temperatures ranging from 15°C to 30°C. Diapause induction was determined by exposing adults to either long or short photoperiods at 20°C and cold tolerance was assessed by exposing adults to 0°C. G. graminea completed development at temperatures ranging from 20°C to 30°C. Linear regression estimated a lower temperature threshold of 11.7°C and 312 degree-days were required to complete development. Diapause was induced when adults were exposed to short photoperiods (10:14 L:D h) at 20°C. The lethal times for diapausing adults of G. graminea at 0°C (LT₅₀?=?19?days, LT₉₀?=?41?days) were two times higher compared to Gratiana boliviana Spaeth, a biological control agent already established in south and central Florida, USA. The presence of diapause and the greater cold tolerance suggest that G. graminea may establish and perform better than G. boliviana in northern Florida.     

Flow cytometric analysis from fish samples stored at low,ultra-low and cryogenic temperatures

Flow cytometry is a valuable tool in biomedical and animal sciences. However, equipment used for such analysis presents limitations at field conditions, suggesting then preservation procedures for future analysis at laboratory conditions. In this study, freezing at low (−20 °C), ultra-low (−80 °C) and cryogenic temperatures (−196 °C, i.e. liquid nitrogen) were used as preservation procedures of fish tissue. Samples were maintained in 0.9% NaCl or lysing solution, and stored at the temperatures above for 0 (fresh control), 60, 120 and 180 days of storage. After storage, the samples were thawed and proceeded to flow cytometric analysis. Storage at low temperatures (−20 °C), both in lysing and 0.9% NaCl, exhibited poor results when analyzed after 60, 120 and 180 days, showing noisy peaks, deviation in the DNA content and absence of peaks. Ultralow (−80 °C) and cryogenic (−196 °C) temperatures, both in lysing solution and 0.9% NaCl, showed good results and high quality of histograms. Both storage procedures gave similar histograms and DNA content in comparison with control group (fresh) even after 60, 120 and 180 days of storage, exhibiting the main peak at 2C content from diploid cells and a secondary peak at 4C derived from dividing cells. In conclusion, samples may be stored for 180 days at −80 °C and −196 °C in both, 0.9% NaCl or lysing solution. As cryogenic temperatures in liquid nitrogen permits indefinite storage, this procedure should be used for long-term preservation.     

Survival of Cornus sericea L. stem cortical cells following immersion in liquid helium

Abstract Cold-acclimated stems of red-osier dogwood (Cornus sericea L.) were sampled in midwinter and early spring and subjected to the following low temperature treatments: (a)0 →?40 → 0°C; (b) 0 →?40 →? 196 → 0°C; (c) 0 →?40 →?196 →?269 →?196 → 0°C; (d) 0 →?40 →?269 →?196 → 0°C; (e) 0 →?196 → 0°C; (f) 0 →?269 →?196 →0°C. The cortical parenchyma cells of the outer stem layers survived exposure to ?269°C when pre-frozen to ?40°C and either transferred directly to ?269°C or to ?196°C and then to ?269°C (treatments c and d). Acclimated stems transferred to a greenhouse (22°C) 2 weeks prior to the low temperature treatments deacclimated and were not able to survive freezing to ?10°C. Cortical cells of stem samples taken in March, near the time when dogwood naturally deacclimates, survived ?196°C (treatment b), but not ?269°C (treatment cord). Thus, the freezing tolerance of dogwood varies seasonally from near ?10°C to below ?269°C.     

Optimal temperature ranges for control of cooling rate.

Survival of hamster fibroblasts following cooling at 1 °C/min to various subzero temperatures in the presence of penetrating or nonpenetrating cryoprotective agents was examined. In the presence of nonpenetrating agents maximum recovery was obtained when the cooling rate was controlled between ?5 and ?20 °C followed by rapid cooling to ?196 °C. For penetrating agents recovery was maximal in samples cooled at 1 °C/min to ?30 °C or lower. These different temperature ranges for maximum recovery indicate different modes of actions of penetrating and nonpenetrating cryoprotective agents. The action of penetrating agents appear to be based on their colligative properties. Nonpenetrating agents may promote electrolyte leaks out of the cell and a corresponding osmotic efflux of cell water during slow cooling, thereby reducing the amount of intracellular ice present at ?196 °C.     

Rooster spermatozoan motility,forward progression,and fertility after storage at 25, 5, or −196 °C in various extenders

Rooster spermatozoa were stored at 25, 5, or ?196 °C in either TC199, a pyruvate-lactate mouse ova culture medium, or as undiluted semen. There was a linear decrease in percent of motile sperm during storage at 25 or 5 °C in all cases, and a curvilinear decrease with increasing storage times at ?196 °C. Percent of motile sperm present after increasing storage time suggested pyruvate-lactate is a better extender than TC199 at the three storage temperatures studied. Pullets inseminated with 1 × 10⁸ motile sperm using fresh sperm diluted in TC199 or pyruvate-lactate, or stored 24 hr at 5 or ?196 °C produced 68.7, 74.1, 20.6, and 10.8% fertile eggs, respectively. The differences in fertility between controls or between samples stored at 5 and ?196 °C were not significant. However, fertility from sperm stored at 5 and ?196 °C was significantly lower (p < .05) than both control groups. Thus, it can be concluded that TC199 or pyruvate-lactate may be used to dilute fresh rooster semen collections prior to insemination. In contrast, fertility of rooster sperm is not satisfactorily maintained after 5 or ?196 °C storage for 24 hr in a pyruvate-lactate extender.     

Proteomics of Colwellia psychrerythraea at subzero temperatures – a life with limited movement,flexible membranes and vital DNA repair

The mechanisms that allow psychrophilic bacteria to remain metabolically active at subzero temperatures result from form and function of their proteins. We present first proteomic evidence of physiological changes of the marine psychrophile Colwellia psychrerythraea 34H (Cp34H) after exposure to subzero temperatures (?1, and ?10°C in ice) through 8 weeks. Protein abundance was compared between different treatments to understand the effects of temperature and time, independently and jointly, within cells transitioning to, and being maintained in ice. Parallel [3H]‐leucine and [3H]–thymidine incubations indicated active protein and DNA synthesis to ?10°C. Mass spectrometry‐based proteomics identified 1763 proteins across four experimental treatments. Proteins involved in osmolyte regulation and polymer secretion were found constitutively present across all treatments, suggesting that they are required for metabolic success below 0°C. Differentially abundant protein groups indicated a reallocation of resources from DNA binding to DNA repair and from motility to chemo‐taxis and sensing. Changes to iron and nitrogen metabolism, cellular membrane structures, and protein synthesis and folding were also revealed. By elucidating vital strategies during life in ice, this study provides novel insight into the extensive molecular adaptations that occur in cold‐adapted marine organisms to sustain cellular function in their habitat.     

Visualization of freezing damage. II. Structural alterations during warming

There is a growing amount of indirect evidence which suggests that the loss in viability of rapidly cooled cells is due to recrystallization of intracellular ice. This possibility was tested by an evaluation of the formation of morphological artifacts in rapidly cooled cells to determine whether this process can account for the loss in viability. Samples of the common yeast Saccharomyces cerevisiae were frozen at 1.8 or 1500 °C/min, and the structure of the frozen cells was examined by the use of freeze-fracturing techniques. Other cells cooled at the same rate were warmed to temperatures ranging from ?20 ° to ?50 °C and then rapidly cooled to ?196 °C, a procedure that should cause small ice crystals to coalesce by the process of migratory recrystallization. Cells cooled at 1500 °C/min and then warmed to temperatures above ?40 °C formed large intracellular ice crystals within 30 min, and appreciable recrystallization occurred at temperatures as low as ?45 °C. Cells cooled at 1.8 °C/min and warmed to temperatures as high as ?20 °C underwent little structural alteration. These results demonstrate that intracellular ice can cause morphological artifacts. The correlation between the temperature at which rapid recrystallization begins and the temperature at which the cells are inactivated indicates that recrystallization is responsible for the death of rapidly cooled cells.     

Studies of l(3)c43hs1 a polyphasic, temperature-sensitive mutant of Drosophila melanogaster with a variety of imaginal disc defects.

The heat-sensitive, lethal mutation l(3)c43^hs1 (3–49.0) produces wide variety of defects in the imaginal discs of Drosophila melanogaster. At permissive temperatures (20°C or lower), homozygotes are viable, but sterile. At 22°C, lethality occurs during the late pupal stage, and at 25°C or higher, lethality occurs during the third larval instar. The imaginal-disc abnormalities observed after exposure to restrictive temperatures include: deficiencies of head structures, duplications and deficiencies of the antenna, a homeotic transformation of the arista to tarsus, duplications and deficiencies of wing and haltere structures, differentiation of amorphous cuticular material in the wing blade, an increase in the number of sex-comb teeth, and disruption of the normal segmentation of the tarsus. Exposure to 27°C for 24 hr at different times in the life cycle revealed that each of these defects has a characteristic temperature-sensitive period (TSP) during the larval stages. Injection of wing discs before and after their TSP showed that the mutation is expressed autonomously. These results are discussed in relation to the role that the l(3)c43⁺ gene plays in the development of imaginal discs.     

Viability and Morphology of rat heart cells after freezing and thawing of the whole heart

Intact adult rat hearts were cooled in the presence of 10% DMSO according to an external cooling program which approximated the optimal external three-step cooling program for the isolated adult heart cells: 20 min at ?20 °C, 0.2 °C/min from ?20 to ?25, ?30, or ?50 °C, and rapid cooling to ?196 °C. Following rapid thawing, cells were isolated after perfusion with a 0.1% collagenase solution. Only cells which originated from the free wall of the right ventricle could be isolated, even after cooling to ?20 °C. Most cells from hearts cooled to ?196 °C did not survive. When the third cooling step was omitted and the end temperature of the second cooling step was ?30 °C, 38% of the cells excluded trypan blue, 29% were morphologically intact, and 30% showed spontaneous contractions after thawing, expressed as percentages of the control, A much lower survival was found after cooling to ?50 °C.Histological and electron microscopical study of the heart immediately after thawing revealed no differences between hearts cooled to ?20, ?30, or ?196 °C. Also no marked differences were observed between the morphological integrity after freezing and thawing of the atrium, the left and right ventricle walls, and the ventricular septum. The survival data suggest the presence of nonmorphologically detectable alterations in cells frozen to ?196 °C, compared to cells frozen to ?30 °C. The morphological investigations indicate no essential differences in resistance of atrial and ventricular cells to the freezing process.Experiments involving neonatal rat hearts cooled to ?196 °C, according to the method which gave optimal preservation of the isolated cells, revealed that after thawing cells are present from which growing and contracting cultures can be derived. It appears that cells in the neonatal rat heart are more resistant to freezing to ?196 °C than cells in the adult rat heart.     

Effects of high-temperature treatments on development,viability, and heat-shock response in a temperature-sensitive cell-lethal mutant of Drosophila melanogaster

Pulses of various durations at temperatures between 29 and 38°C were applied to developing larvae of Drosophila melanogaster carrying the temperature-sensitive cell-lethal mutation 1 (1)ts726. The results show that it is not possible to reduce the time required for the induction of abnormalities in the mutant by treating larvae with heat pulses at temperatures higher than 29°C. Instead, treatment with high temperature leads to fewer abnormalities than 29°C treatments. Furthermore with high temperature treatments, the mutation has less effect on viability than is seen at 29°C. It is suggested that 1 (1)ts726 leads to abnormalities and death by a temperature-induced imbalance between different physiological or development events, rather than by interfering with the ability of the cell or the organism to withstand high temperature in general.     

Three-step cooling: A preservation method for adult rat heart cells

Adult rat heart cells were exposed to two-step cooling to ?196 °C with different holding periods at different subzero temperatures between both steps. The highest survival based on the percentage of trypan blue-excluding cells was 25% with 10% DMSO and a holding period of 6 min, and 21% with 15% DMSO and a holding period of 30 min. The highest survival based on morphological intactness was about 10%; there was no difference in results after cooling with 10 and 15% DMSO, and after holding between 2 and 30 min. The optimal survival based on the percentage of contracting cells was 52%, with 15% DMSO and a holding period of 2 min.When the holding period was replaced by a programmed cooling stage, the results could be improved. With this threestep cooling method, the optimal values, based on the number of trypan blue-excluding, intact, and contracting cells, were 40, 32, and 60%, respectively. It appeared that in the presence of 10% DMSO, which provided better survival than 5 and 15%, no significantly different results were obtained when the starting temperatures of the second cooling step varied between ?10 and ?20 °C, when the end temperatures varied between ?30 and ?60 °C, or when the cooling rates of the second cooling step varied between 0.1 and 1 °C/min. Three-step cooling provided similar results as linear cooling from 0 to ?100 °C, followed by rapid cooling to ?196 °C.