The influence of metabolic inhibitors and temperature on mutagenic activity during the storage of ethyl methanesulphonate-treated barley seeds

Anaerobic conditions, the inhibitor of aerobic respiration-sodium azide and the inhibitor of proteosynthesis-chloramphenicol did not influence the increase of toxic and mutagenic effects during the storage of ethyl methanesulphonate-treated barley seeds at 15 per cent water content. The storage of ethyl methanesulphonate-treated seeds at ? 20 °C or at 0 °C avoided all after-effects. With increasing storage temperature (5 °C, 15 °C and 25 °C) the increase of injury due to storage was more pronounced.     

Cryopreservation and microencapsulation of a probiotic in alginate-chitosan capsules improves survival in simulated gastrointestinal conditions

The aim of the present study was to focus on the impact of two different methods and the effects of cryoprotectants on the survival of a probiotic bacterium, Streptococcus phocae PI80, during storage. For the protection of freeze dried cells, the optimal storage conditions were determined with a high survival rate. After the freeze drying process, all cryoprotectants exhibited a protective effect on cell viability at all storage temperatures. High relative cell viability was observed when cells were incubated at ?20°C, which was optimum for the protection of S. phocae PI80. Trehalose was the most promising cryoprotectant at all temperatures during the storage period of bacterial cells. The combination of trehalose + skim milk showed more than 85% survivability compared to other combinations at ?20°C for 60 days. In addition, encapsulation of probiotic cells into alginate-chitosan gel capsules showed better survival of S. phocae cells (5.468 ± 0.15 LogCFU/mL) with high bacteriocin activity at ?20°C for six months. The cell-loaded microcapsules remained stable when treated with simulated gastric and intestinal fluids. After 6 h in vivo treatment, the capsules were found to be broken, releasing the probiotic cells directly into the intestinal system of rats. Therefore, microencapsulation was found to be the most efficient technique, which not only protected the cells for a longer time but also released the cells into the in vivo intestinal system.     

The effect of cell concentration on the recovery of human erythrocytes after freezing and thawing in the presence of glycerol

Human erythrocytes, suspended in 2.5 M glycerol in phosphate-buffered saline, were frozen at 35 °C/min to between ?60 and ?65 °C and thawed at 5 °C/min. It was found that cell concentration had a marked effect on cell recovery. When the hematocrit was less than 20%, hemolysis was less than 1% but when the hematocrit exceeded 50%, hemolysis increased, reaching 16% at an hematocrit of 80%. Possible causes of this effect are discussed, and it is suggested that augmentation of solution effects and intracellular freezing may provide a sufficient explanation. The importance of this cell-packing effect for attempts to preserve whole organs is discussed.     

Dynamic motions of ice-binding proteins in living Caenorhabditis elegans using diffracted X-ray blinking and tracking

The dynamic properties of protein molecules are involved in the relationship between their structure and function. Time-resolved X-ray observation enables capturing the structures of biomolecules with picometre-scale precision. However, this technique has yet to be implemented in living animals. Here, we examined diffracted X-ray blinking (DXB) and diffracted X-ray tracking (DXT) to observe the dynamics of a protein located on intestinal cells in adult Caenorhabditis elegans. This in vivo tissue-specific DXB was examined at temperatures from 20 °C to ?10 °C for a recombinant ice-binding protein from Antarctomyces psychrotrophicus (AnpIBP) connected with the cells through a transmembrane CD4 protein equipped with a glycine-serine linker. AnpIBP inhibits ice growth at subzero temperatures by binding to ice crystals. We found that the rotational motion of AnpIBP decreases at ?10 °C. In contrast, the motion of the AnpIBP mutant, which has a defective ice-binding ability, did not decrease at ?10 °C. The twisting and tilting motional speeds of AnpIBPs measured above 5 °C by DXT were always higher than those of the defective AnpIBP mutant. These results suggest that wild-type AnpIBP is highly mobile in solution, and it is halted at subzero temperatures through ice binding. DXB and DXT allow for exploring protein behaviour in live animals with subnano resolution precision.     

Reversible filamentous growth in the psychrophile Bacillus psychrophilus

At the near-maximum growth temperature of 32.5 °C, the psychrophile Bacillus psychrophilus loses the ability to septate and divide, resulting in the formation of filaments, which are four to six times longer than cells grown at 20 °C. DNA synthesis relative to growth occurs at the same rate both in the filaments at 32.5 °C, (which actually become multi-nucleated) and in normal-size cells at 20 °C, showing that the inhibition of DNA synthesis by the elevated temperature is not the cause of the filamentous growth, as has been found for other microorganisms. Similarly, temperature-sensitive cell-wall mucopeptide synthesis does not appear to be responsible. Reversal of filament production occurs when preformed filaments are incubated at 20 °C. Such reversal, i.e., septation of preformed filaments, requires the de novo synthesis of protein, probably throughout the reversal period.Filamentous cells are more nutritionally demanding than cells at 20 °C, with at least one substrate becoming limiting within 8 hr at 32.5 °C but not at 20 °C. However, such variation in nutritional requirement is not the cause of filament formation. KCl and NaCl stimulate cell division in cells growing at 32.5 °C but not in preformed filaments. Other membrane-active agents such as lysolecithin, dimethyl sulfoxide, ethanol, sodium oleate, and pantoyl lactone do not stimulate septum formation in filaments.     

Effects of 4-nitro- and 4-sulpho-7-substituted benzofurazans on nucleic acid and protein synthesis of a malignant fibrosarcoma cell line in combination with mild hyperthermia

The inhibitory effects of substituted nitro- and sulphobenzofurazans on DNA, RNA and protein synthesis were compared in a new malignant fibrosarcoma cell line at 37°C and 41°C. The effects of these drugs with and without mild hyperthermia were evaluated by determining the % inhibition of incorporation of ³H-precursors into DNA, RNA and protein. None of the sulphobenzofurazan derivatives (Sbf) were effective inhibitors of nucleic acid and protein synthesis at 37°C nor did they enhance the inhibitory effect of hyperthermia alone. The nitrobenzofurazan derivatives (Nbf) at concentrations 10% that used for the Sbf derivatives strongly inhibited biopolymer synthesis in a dose related manner; 4-chloro-7-nitrobenzofurazan (Nbf-Cl) being the most potent inhibitor. Hyperthermia amplified the effect of all the Nbf compounds tested on RNA and protein synthesis but did not further affect DNA synthesis. This selective synergistic effect was most pronounced when the lowest concentrations of Nbf compounds were studied. The synergism however, did not follow a uniform pattern. 6-Mercaptopurine and 6-(1-methyl-4-nitro-5-imidazoyl)thiopurine (Azathioprine) (100 μM) had marginal effects on nucleic acid and protein synthesis when the cells were exposed to these two thiopurines for 1 h at both 37°C and 41°C and they had only a moderate inhibitory effect after exposure for 15 h.     

Effect of storage temperature on gentamicin release from antibiotic-coated bone chips

Freezing is the most common method for storing bones until use in skeletal reconstruction. However, the effect of freezing on antibiotic delivery from antibiotic-coated bone has not been evaluated. In this study, we compared antibiotic delivery in vitro from gentamicin-coated human bone stored at different temperatures. Bone chips obtained from human femur heads were chemically cleaned and mixed with gentamicin sulfate. Samples were stored for 4 months at ?20 °C, 4 months at ?80 °C, or evaluated immediately without freezing. Antibiotic release from the bone chips was measured using Bacillus subtilis as an indicator strain. Zones of inhibition and rates of gentamicin release were similar in all three groups. Storage at ?20 and ?80 °C for bone allografts has no effect on gentamicin release from chemically cleaned bone chips.     

Acute Toxicity of Arsenic under Different Temperatures and Salinity Conditions on the White Shrimp Litopenaeus vannamei

The aim of this study was to determine acute toxicity in the post larvae of the white shrimp Litopenaeus vannamei after 96 h of exposure to dissolved arsenic under three different temperatures and salinity conditions. Recent reports have shown an increase in the presence of this metalloid in coastal waters, estuaries, and lagoons along the Mexican coast. The white shrimp stands out for its adaptability to temperature and salinity changes and for being the main product for many commercial fisheries; it has the highest volume of oceanic capture and production in Mexican shrimp farms. Lethal concentrations (LC₅₀–96 h) were obtained at nine different combinations (3?×?3 combinations in total) of temperature (20, 25, and 30 °C) and salinity (17, 25, and 33) showing mean LC₅₀–96 h values (±standard error) of 9.13?±?0.76, 9.17?±?0.56, and 6.23?±?0.57 mgAs?L^?1(at 20 °C and 17, 25, and 33 salinity); 12.29?±?2.09, 8.70?±?0.82, and 8.03?±?0.59 mgAs?L^?1 (at 25 °C and 17, 25, and 33 salinity); and 7.84?±?1.30, 8.49?±?1.40, and 7.54?±?0.51 mgAs?L^?1 (at 30 °C and 17, 25, and 33 salinity), respectively. No significant differences were observed for the optimal temperature and isosmotic point of maintenance (25 °C–S 25) for the species, with respect to the other experimental conditions tested, except for at 20 °C–S 33, which was the most toxic. Toxicity under 20 °C–S 33 conditions was also higher than 25 °C–S 17 and 20 °C (S 17 or 25). The least toxic condition was 25 °C–S 17. All this suggests that the toxic effect of arsenic is not affected by temperature changes; it depends on the osmoregulatory pattern developed by the shrimp, either hyperosmotic at low salinity or hiposmotic at high salinity, as observed at least on the extreme salinity conditions here tested (17 and 33). However, further studies testing salinities near the isosmotic point (between 20 and 30 salinities) are needed to clarify these mechanisms.     

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.     

Regeneration of Plants from Cell Suspensions Frozen at −20, −70 and −196°C

Cell suspensions of carrot, Datura, tobacco and soybean subjected to ?20°C, ?70°C and ?196°C in the presence of a suitable cryoprotective agent, and stored for various lengths of time have been revived. After revival these cells divided to form callus masses. Direct immersion in liquid nitrogen invariably killed the cells, whereas cooling at the rate of 1 or 2°C/min, or pre-freezing briefly at ?20 and ?70°C, followed by freezing at ?196°C retained the viability. Depending on the plant species up to 70% of the cell clumps could withstand ultra-cooling. Tobacco and Datura cell suspensions were more sensitive to cold treatment than were those of carrot. Actively growing cell suspensions containing small cell-clumps revived rapidly, while filtered cell-suspensions of free cells only occasionally survived. Calli of tobacco and carrot obtained from frozen suspensions have been regenerated into plants.     

Sensitivity of pigmented mucosa and skin to freezing injury.

Pigmented areas of canine skin and oral mucosa were subjected to freezing in situ at various temperatures for the purpose of investigating possible differences in the sensitivity of epidermal cells to cold injury. Destruction of melanocytes occurred in the range of ?4 to ?7 °C, while squamous cells resisted injury even at ?20 °C. Replacement of the lost pigmented cells occurred from the normal tissue at the periphery of the injured area. The experiments suggest that selective destruction of pigmented lesions in clinical conditions may be achieved by freezing tissue to ?4 to ?7 °C or colder, but not to exceed ?20 °C in order to avoid destruction of squamous cells. The experiments also support wider use of cryosurgery for pigmented lesions of the skin and oral cavity.     

The mechanism of cryohemolysis: by direct observation with the cryomicroscope and the electron microscope.

Blood films (3–8 μm thick) supported between two glass coverslips were frozen to ?20 °C. In the extracellular areas, ice cavities of the order of 0.2 μm separated by bands of dense plasma were evident when examined with the electron microscope; intracellular ice was not observed with the light microscope. Electron microscopy also showed the presence of intracellular ice particles of the order of 0.2–0.7 μm, these appeared as fine reticulations when observed with the light microscope. Upon gradual rewarming the following changes were observed: recrystallization in the extracellular matrix (?18 to ?8 °C), intracellular recrystallization (?13 to ?10 °C), transfer of water from erythrocytes to extracellular areas (?9 to ?7 °C), and melting and hemolysis (?6 to ?2 °C).Freezing of blood at ?3 °C and subsequent thawing did not cause hemolysis of the red cells. In blood frozen at ?3 °C and cooled to ?20 °C or frozen by abrupt exposure to 20 °C the erythrocytes hemolyzed in 7/16–11/16 of a second, whereas in blood frozen at ?3 °C and cooled to ?10 °C the cells hemolyzed in 5–15 sec even though the mode if lysis (i.e., uniform seepage of hemoglobin from the surface of the cell) was similar in all cases. This indicates that the presence of intracellular ice does not seem to play a major role in the injury to the erythrocytes. The mechanism of cryoinjury demonstrated by hemolysis has been discussed.     

Thermolabile analogue proteins and the entry of HeLa and CHO cells into division

Entry of HeLa and CHO-10 cells into mitosis can be inhibited by incorporation of p-fluorophenylalanine at certain temperatures, 37 °C for the former cell type and 39.5 °C for the latter. At lower temperatures, 32 °C in the former and 37 °C in the latter, the analogue does not inhibit entry of cells into mitosis. The possibility that the analogue is not incorporated at the permissive temperatures has been ruled out; indeed incorporation is relatively greater at the permissive temperatures. The results suggest that the physiological properties of analogue protein molecules differ depending on the temperature at which they are synthesized; the higher the temperature the more likely they are to malfunction.     

Viability of plum ovules at different temperatures

The viability of ovules was studied in five plum cultivars under laboratory conditions at four constant temperatures: 5°C, 10°C, 15°C and 20°C and under field conditions over two years. During 10 days from the onset of full bloom, ovule viability in cvs ‘?a?anska Rana’, ‘?a?anska Najbolja’ and ‘?a?anska Lepotica’ was between 80–100 % at the temperatures of 5°C, 10°C and 15°C, in both years. In the same period, ovule viability in cvs ‘Wangenheims Frühzwetsche’ and ‘Po?ega?a’ was lower, but never below 50%. At the constant temperature of 20°C, all plum cultivars showed a decline in longevity of ovule viability, which was pronounced in cv. ‘?a?anska Rana’. During the 10 days from the onset of full bloom, ovule viability in all five plum cultivars under field conditions showed a high viability, which approximated to the ovule viability of the cultivars at the constant temperatures of 5°C, 10°C and 15°C, in both years. Determination of the longevity of ovule viability in the mentioned plum cultivars is of great importance due to its effect on the effective pollination period and fertilisation success. This paper deals in detail with the interrelations between the temperature effects on ovule viability, pollen tube growth and fertilisation, as well as on fruit setting.     

Increased production of PGI2-like activity by frozen-thawed rat aorta

The effects of storage conditions, temperature, and time on the ability of the rat thoracic aorta to produce a platelet aggregation inhibitor were investigated. Aortic fragments were incubated in Tris buffer, aliquots of which were then tested for their ability to inhibit ADP-induced human platelet aggregation. The incubation fluid of samples that had been soaked in Tris buffer at 4°C for 24 hours contained no inhibitor activity, whereas the incubation fluid of similar samples that had been kept at 4°C but not soaked in buffer contained comparable inhibitor activity as that of fresh samples. The incubation fluid of samples that had been kept at ?20°C or ?80°C contained greater inhibitor activity than that of fresh samples, and was maintained in ?20°C samples for 7 days, and ?80° samples for 28 days. The aortic inhibitor had similar properties as PGI₂.     

Development of Geocoris varius and G. proteus (Hemiptera: Geocoridae) provided with Ephestia kuehniella (Lepidoptera: Pyralidae) eggs

We studied the development of Geocoris varius (Uhler) and Geocoris proteus Distant reared on Ephestia kuehniella Zeller eggs at 20, 24, 26, 30, 33, or 36?°C. The lower developmental thresholds (T ₀) and the thermal constants (K) of eggs and nymphs of G. varius were 13.3?°C, 151.1 degree-days and 13.4?°C, 433.0 degree-days, respectively; those of G. proteus were 16.1?°C, 98.3 degree-days and 16.9?°C, 226.9 degree-days, respectively. The hatch rate of G. varius eggs was significantly lower at 33?°C than at ??30?°C, and no eggs hatched at 36?°C. That of G. proteus was lowest at 20?°C and did not decline significantly at 36?°C. The survival rate throughout the nymphal period increased with temperature up to 30?°C in G. varius, and it was lowest at 20?°C in G. proteus. Thus, the optimal rearing temperatures for immature stages appear to be about 24?C30?°C for G. varius and 26?C33?°C for G. proteus. It might be possible to improve the efficiency of their mass production by controlling the rearing temperature in the above ranges. This would also make the developmental stages of nymphs more uniform and so prevent cannibalism in mass rearing.     

Yeasts preservation: alternatives for lyophilisation

The aim of the study was to compare the effect of two low-cost, low technology traditional methods for drying starter cultures with standard lyophilisation. Lyophilised yeast cultures and yeast cultures preserved in dry rice cakes and dry plant fibre strands were examined for viable cell counts during 6?months storage at 4 and 25?°C. None of the yeast cultures showed a significant loss in viable cell count during 6?months of storage at 4?°C upon lyophilisation and preservation in dry rice cakes. During storage at 25?°C in the dark, yeast cultures preserved in dry rice cakes, and lyophilised cultures of Saccharomyces cerevisiae and Issatchenkia orientalis showed no significant loss of viable cells up to 4?months of storage. Yeast cultures preserved in dry plant fibre strands had the greatest loss of viable count during the 6?months of storage at 25?°C. Preservation of yeasts cultures in dry rice cakes provided better survival during storage at 4?°C than lyophilisation. The current study demonstrated that traditional methods can be useful and effective for starter culture preservation in small-scale, low-tech applications.