Induction and decay of thermotolerance in rainbow trout fibroblasts

Thermotolerance was studied in the rainbow trout fibroblast cell line RTG-2. RTG-2 cultures that had been incubated at 28 degrees C for 24 h were better able to withstand ultimately lethal temperatures above 28 degrees C than RTG-2 cultures that had been maintained at the routine growth temperature of 22 degrees C. This thermotolerance developed rapidly between 3 and 6 h and was fully developed by 24 h at 28 degrees C. After development for 24 hr at 28 degrees C, thermotolerance showed little change over 72 h at 0 and 5 degrees C but approximately a 40 and 60% reduction at 10 and 22 degrees C, respectively. This is the first demonstration of heat-induced thermal resistance in the cells of a poikilothermic vertebrate.     

Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor

Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below 32°C resulted in growth suppression. However, specific productivity of FSH, q _FSH, increased as culture temperature decreased, and the maximum q _FSH of 43.4 ng/10⁶ cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures, we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 10⁷ cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising.     

Maintaining mosquito cell lines at high tempetatures: Effects on the replication of flaviviruses

Summary The upper thermal limit for maintenance of eleven mosquito cell lines was studied. Although most cell lines could be grown at 32°C to 34°C,Anopheles stephensi cell line could be maintained at 37°C. At higher temperatures initial growth rate was higher, but yield of cells after about a week of incubation was lower than at the standard temperature (28°C). Replication of several flaviviruses inAedes albopictus cell cultures adapted to 34.5°C was faster, and viral titers were higher than at 28°C.     

SURVIVAL OF SCENEDESMUS ACUMINATUS (CHLOROPHYCEAE) IN DARKNESS1

Survival of the green alga Scenedesmus acuminatus Lagerh. in complete darkness was studied in axenic batch cultures at 7°C and 22°C for three months. The decrease in cell numbers was insensitive to temperature and slower than the loss of dry weight. However, the lag phase before cells began to lyse was more than twice as long at 7° C than at 22°C. The decline in cellular carbohydrates and proteins occurred in two phases. During the first 3-4 days, the decrease in cellular carbohydrate levels was significantly accelerated and temperature-sensitive. Pyrenoids disappeared within 5 days of darkness. Proteins showed 20-fold higher degradation rates at 22°C than at 7°C during the first 4 days. Thereafter, the rates of carbohydrate and protein decomposition were slow and temperature-independent. By contrast, lipids degraded only little at virtually constant and temperature-insensitive rates over the entire experimental period. After three months of dark incubation, about 40% of the remaining cells had retained their growth potential. However, the lag phase, after which cell division was resumed when exposed to light, increased with the duration of the previous dark period. The decrease in photo synthetic potential, which was more pronounced at 22° C than at 7° C, was apparent both in declining maximum assimilation numbers and maximum quantum yields. Cellular chlorophyll a concentrations in surviving cells decreased only slightly. We conclude that the primary means by which S. acuminatus survives extended dark periods is by reduction of catabolic reactions. This was suggested by the slow loss of cell weight. No evidence of significant heterotrophic acetate uptake was found. The initial temperature-dependence of most observed processes indicates that in natural environments chances for survival of algae are augmented by the prevailing low water temperatures.     

Kinetic studies of benzpyrene and hydroxybenzpyrene metabolism.

The kinetics of benzypyrene (BP) metabolism were examined in liver microsomes, and in accordance with the results of Hansen and Fouts [9] exhibited curcilinear Lineweaver-Burk plots. The problem was exacerbated in microsomes of 3-methylcholanthrene (MC-ms) treated rats. The Km for BP, measuring hydroxybenzypyrene (OHBP) appearance was about 0.3 muM in MC-treated adult rats and about 1.0 muM in untreated rats. These values were obtained using a substrate range of 0.2-2.0 muM benzpyrene, 20 mug of microsomal protein/ml and a 3 min assay time. With longer assay times and with higher microsomal protein concentrations curvilinear reciprocal plots were obtained. This was found to be due to a combination of three factors, namely non-specific binding of BP to the microsomes, rapid depletion of substrate, and further metabolism of hydroxy products. At 100 mug microsomal protein/ml about 50% of added BP was non-specifically bound to the microsomes in the range of 0.2-2.0 muM BP. Addition of albumin to the medium (1 mg/ml) greatly enhanced the BP hydroxylase activity but only slightly increased the amount of BP remaining in the medium after sedimentation of the microsomes by centrifugation. 3-OHBP, one of the phenolic products of BP metabolism was found to be metabolized to a non-fluorescent products(s); the Km for this compound was similar to that for BP. Differences were seen in the Vmax rates of BP disappearance and OHBP appearance. Disappearance of BP is several fold faster than OHBP appearance and has a larger Km. The latter may be due to the need to use higher amounts of protein and to allow depletion of enough substrate to make measurements significantly reproducible or the higher Km may reflect a composite value for different routes of BP metabolism.     

Conditionally lethal mutations in Chinese hamster cells. I. Isolation of a temperature-sensitive line and its investigation by cell cycle studies

The isolation of a temperature sensitive cell line from the Chinese hamster line CCL39 of the American Type Culture Collection is described. At the nonpermissive temperature (39°C) the cells become attached to the surface of tissue culture dishes, but no microscopically observable colonies are formed upon prolonged incubation. Exposure to the high temperature for more than 24 hours leads to an almost complete loss in viability. A karyotypic analysis showed that this new line has lost one of the medium-sized metacentric chromosomes, although no proof is available so far to show that this loss is not simply coincidental. In nonsynchronized cultures transferred to 39°C DNA synthesis stops first, RNA synthesis shortly thereafter, while protein synthesis (turnover) continues for a longer time. After such a shift the cell number increases by less than 15% as measured with the Coulter counter. Studies with synchronized cultures give the following results: (1) one round of DNA synthesis can occur at 39°C when the cells are released from serum starvation or a hydroxyurea block, or when mitotic cells are placed at 39°C; (2) the entry of cells into metaphase of mitosis at 39°C is almost normal when the preceding time interval at 39°C is only eight hours (release of cells from G₁/S boundary), but considerably reduced when the cells spend an additional 12 to 15 hours at 39°C in G₁ (release from serum starvation). Infection by SV40 virus temporarily induces DNA synthesis after it has come to a stop at the nonpermissive temperature, but cells permanently transformed by SV40 still exhibit the temperature-sensitive phenotype.     

Neurotensin and bombesin: differential effects on body temperature of mice after intracisternal administration

Intracisternal administration of neurotensin or bombesin produces a significant hypothermic response in rodents in an ambient temperature of 23°C or below; bombesin has been reported to produce a significant hyperthermic response in rats at 36°C, but no change in colonic temperature at ambient temperatures between 31 and 33°C. In this study we compared the effects of the two neuropeptides on colonic temperature of mice exposed to different ambient temperatures to determine whether neurotensin also produces a poikilothermic state. From a series of experiments conducted at ambient temperatures of 4, 23, 26, 30, 34 and 38°C, in which mice received an intracisternal injection of an equimolar dose (0.6 nmol) of neurotensin or bombesin (or vehicle), we noted that the two neuropeptides produce different effects on colonic temperature. At ambient temperatures of 26°C and below, both neurotensin and bombesin produce a significant hypothermic response; however, at higher temperatures bombesin has no effect (30°C) or produces hyperthermia (34°C). In contrast, neurotensin produces hypothermia at 30°C and no significant effect at 34 and 38°C. In addition, a wide range of doses of neurotensin failed to produce the poikilothermic effects characteristic of centrally administered bombesin.     

Characterization of a reptilian epithelioid skin cell line derived from the green sea turtle,Chelonia mydas

Summary A continuous line of epithelioid cells was established from explant skin tissues of the green sea turtle,Chelonia mydas. These cells, designated GTS, have been subcultured more than 60 times in commercially available mammalian cell culture medium supplemented with 5% bovine calf serum. Of those temperatures tested, optimal growth was achieved at 30°C although replication occurred between 16 and 37°C. These cells may be held as monolayers at 8°C or stored frozen in growth medium containing 10% dimethylsulfoxide at −70 or −196°C. The modal number of 55 chromosomes per cell is in agreement with the heterogametic female diploid number of this species. The GTS line represents the first established culture of normal epithelioid skin cells to be reported for a poikilothermic species.     

Activation of an Aquareovirus,Chum Salmon Reovirus (CSV), by the Ciliates Tetrahymena thermophila and T. canadensis

For the first time, ciliates have been found to activate rather than inactivate a virus, chum salmon reovirus (CSV). Activation was seen as an increase in viral titre upon incubation of CSV at 22 °C with Tetrahymena canadenesis and two strains of T. thermophila: wild type (B1975) and a temperature conditional mutant for phagocytosis (NP1). The titre increase was not likely due to replication because CSV had no visible effects on the ciliates and no vertebrate virus has ever been shown unequivocally to replicate in ciliates. When incubated with B1975 and NP1 at 30 °C, CSV was activated only by B1975. Therefore, activation required CSV internalization because at 30 °C only B1975 exhibited phagocytosis. CSV replicated in fish cells at 18 to 26 °C but not at 30 °C. Collectively, these observations point to CSV activation being distinct from replication. Activation is attributed to the CSV capsid being modified in the ciliate phagosomal‐lysosomal system and released in a more infectious form. When allowed to swim in CSV‐infected fish cell cultures, collected, washed, and transferred to uninfected cultures, T. canadensis caused a CSV infection. Overall the results suggest that ciliates could have roles in the environmental dissemination of some fish viral diseases.     

Effects of millimeter-wave radiation on monolayer cell cultures. II. Scanning and transmission electron microscopy

Both thermal and athermal effects of millimeter-wave radiation on BHK-21/C13 cells were sought using scanning and transmission electron microscopy in conjunction with an in vitro technique that allows direct exposure of monolayer cultures to high average power densities. Culture dishes were irradiated by placing them on the open end of an E- or U-band wave guide. This technique exposes different regions of the cell monolayer lying along the longer axis of the wave guide aperture to varying power densities ranging from zero at each edge to twice the average power density at the center. Cell ultrastructure was unaffected by microwave radiation for 1 hour (41.8 or 74.0 GHz, average power densitites = 320 or 450 mW/cm², respectively) with or without cooling by rapid recirculation of the culture medium. Temperature in recirculated cultures was held at 37.2 °C, and that in noncooled cultures never exceeded 42 °C during irradiation at either power density. In contrast, cell morphology was affected by microwave exposure whenever irradiation conditions were altered so that the temperature of the monolayer reached or exceeded 44.5 °C. Ultrastructural alterations included breakage of cell processes, progressive detachment of cells from the substrate, increased clumping of heterochromatin in the nuclei, and the appearance of large empty vesicles in the cytoplasm. Such morphological changes resulted from either application of higher average power densities or irradiation at the power densities described above at a higher ambient temperature (>38.5°C).     

Metabolic and functional characteristics of erythrocytes from Glycera and Noetia

• 1.1. Annelid and molluscan red blood cells (RBC) may de differentiated metabolically from vertebrate RBC by their increased permeability to substrate, their magnitude of amino acid catabolism and their higher aerobic metabolism.
• 2.2. At 22°C, Glycera and Noetia RBC oxidize glucose and glutamate to CO₂ without accumulation of either d- or l-lactate. By comparison, the oxidation of glutamate by rat and chicken RBC is negligible at this temperature despite its incorporation into the cells.
• 3.3. At 37°C, chicken RBC oxidize glutamate at a rate 4 times greater than at 22°C, with oxygen uptake still lower than that in Noetia RBC at 32°C. At 37°C, rat RBC do not increase their oxidation of glutamate above that at 22°C, but oxygen uptake increases to slightly more than half that of chicken RBC.
• 4.4. Our finclings indicate that RBC of these two invertebrate species have both a higher aerobic metabolism and lower anerobic capacity than vertebrate RBC.
• 5.5. Moreover, the annelid and molluscan RBC have a relatively lower activity of the pentose phosphate (PPO₄) pathway than vertebrate RBC, as evidenced by their higher thermal sensitivity of oxygen uptake and their higher *C₁O₂/*C₆O₂ isotope ratio.

     

Morphological Changes of <Emphasis Type="Italic">Pseudomonas pseudoalcaligenes</Emphasis> in Response to Temperature Selection

Adaptation to novel environments usually entails morphological changes. The cell morphology of six experimental populations of Pseudomonas pseudoalcaligenes and their common ancestor were examined with scanning electron microscopy (SEM). The six experimental populations were propagated under different temperatures for 10 months: three of them cultured at constant normal temperature (35°C) forming the control group, and the other three cultured at incremental higher temperatures (from 41° to 47°C) as the HT group. SEM showed the deformed and elongated cells in the 6-h cultures of both ancestral and control populations at 45°C, indicating that 45°C is stressful for the ancestral and the control populations. In contrast, the HT populations retained normal cell shape in the 6-h cultures at both 35°C and 45°C. The mean cell volumes of control and HT populations increased 29% and 34%, respectively, relative to the ancestor at their respective thermal regimens, suggestion that the culturing conditions might favor larger cells. Received: 27 March 2002 / Accepted: 30 April 2002     

DNA synthesis and mitosis in a temperature sensitive Chinese hamster cell line

Viability, DNA synthesis and mitosis have been followed in the temperature sensitive Chinese hamster cell mutant K12 under permissive and non-permissive conditions. On incubation at 40°C cells retained their ability to form colonies at 33°C for 15 to 20 hours, but viability was lost gradually during the following 20 hours. When random cultures of K12 were shifted to 40°C the rate of DNA synthesis was normal for three to four hours but then decreased markedly, reaching 95% inhibition after 24 hours. Under the same conditions mitosis was inhibited after 15 hours. If cultures which had been incubated at 40°C for 16 hours were placed at 33°C the rate of DNA synthesis increased five hours after the shift down and mitosis 18 hours after. These results can be interpreted on the assumption that K12 at 40°C is unable to complete a step in the cell cycle which is essential for DNA synthesis and which occurs three to four hours before the start of S at 33°C.     

Metabolic differences inBacillus stearothermophilus grown at 55°C and 37°C

Summary Bacillus stearothermophilus was adapted to grow at 55°C and 37°C in a complex medium with almost equivalent yields in cell mass. In both temperature ranges the maximum specific growth rates (μ_max) were identical. Cellular extracts of this bacterium showed remarkable differences in the activity levels of several enzymes, depending on the respective growth temperature. High activities of glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase were observed in bacteria from thermophilic cultures (55°C) and the respiratory quotient exceeded 1.0. Under anaerobic conditions at 55°C μ_max was the same as in aerobic cultures. No alcohol dehydrogenase was detected in cells from mesophilic cultures (37°C), however, and the level of glyceraldehyde-3-phosphate dehydrogenase was also extremely low under mesophilic conditions. Succinate dehydrogenase and isocitrate dehydrogenase activity appeared to be higher in bacteria grown at 37°C; the resspiratory quotient was always lower than 1.0. At 37°C, acetoin formation was observed regularly, a fermentation product which was never detected in 55°C-cultures. Under anaerobic conditions at 37°C a very low growth rate was found. When adapted to grow at 37°C or 55°C,B. stearothermophilus is apparently able to use different catabolic systems.     

Effects of hypothermia on the survival and cryopreservation of minipig ileal cells and Chinese hamster ovary cells.

Temperature of culture can be used to modulate cellular metabolism for improving small intestinal cell culture and cryopreservation. An hypothermia pretreatment (2 days at 25°C and 3 hours recovery at 37°C) improved hamster cell survival to freeze-thaw damage (p < 0.01) but decreased the survival of 2 immortal pig ileal cell lines even though epithelioid IPI-2I cells were more tolerant to hypothermia than IPI-I fibroblasts. Epithelioid cells survived 3 days at 25°C with unaltered expression of cytokeratin-18 whereas colonies of fibroblasts did not survive more than a day at 25°C (p < 0.001). These results suggest that hypothermia-tolerance of pig ileal cell lines might differ according to cell lineage calling for further experiments on small intestinal primary cell culture.     

Establishment of a variant cell clone growing at 41° C from embryonic rat and tupaia (tree shrew) fibroblast cells

Summary Rat and tupaia 41° C temperature variant cell clones were derived from parental embryonic cells, cloned and established in tissue cultures. Both variant cell clones grew permanently at 41° C. The morphology of these cell clones was altered in comparison to the original fibroblast cell clones. The cell biological characterization of the rat and tupaia 41° C temperature variant cell clones showed that both cell clones were stable. After abolishing the selection pressure (incubation at 41° C) for more than 10 further cell passages by incubation at 37°C and then raising the temperature again to 41° C, neither of the cell clones lost their newly acquired property of prowing at 41° C. This fact demonstrates that the newly acquired property is certain to be genetically manifest in both cell clones. The modal number of chromosomes of the rat 41° C temperature variant cell clone was increased, and the case of the tupaia variant cell clone, bimodality was observed. The plating efficiency of both cell clones did not rise significantly in comparison to the parental cells. Neither of the 41° C temperature variant cell clones grew in semi-solid medium. This work was partially supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 136.     

Thymidine nucleotide synthesis and catabolism by CHO cells and their changes during the cell cycle

At 0°C, CHO cells efficiently incorporated [³H]thymidine into the nucleotide fraction, but not into DNA. Upon reincubation of asynchronous cultures at 37°C, 15–25% of the radioactivity contained in the cellular nucleotide fraction was released, in the form of thymidine, into the culture medium. At 0°C, however, radioactivity of the nucleotide fraction was retained within the cells. Similarly, dTMP phosphatase (EC 3.1.3.35) in cell extracts was active at 37°C, but not at 0°C, whereas thymidine kinase (EC 2.7.1.21) was active at both temperatures. If synchronous cultures in Gl phase were prelabeled at 0°C and reincubated at 37°C, almost all radioactivity in the nucleotide fraction was released into the medium, whereas in S-phase cultures nearly all radioactivity of the nucleotide fraction was incorporated into DNA. In synchronous S-phase cultures treated with hydroxyurea, radioactivity in the nucleotide fraction was released into the medium at a rate considerably lower than that observed for Gl-phase cells. Rates of endogenous synthesis of thymidine nucleotides were calculated from changes of cellular thymidine nucleotide content, incorporation of thymidine nucleotides into DNA and release of thymidine into the medium during reincubation of prelabeled cultures in thymidine-free medium. The results obtained (see Table III) reveal marked differences between Gl and S phases with respect to the determinants of thymidine nucleotide metabolism.     

Differential metabolic response of cultured rice (Oryza sativa) cells exposed to high‐ and low‐temperature stress

Global mean temperatures are expected to rise by 2–4.5°C by 2100, accompanied by an increase in frequency and amplitude of extreme temperature events. Greater climatic extremes and an expanded range of cultivation will expose rice to increasing stress in the future. Understanding gene expression in disparate thermal regimes is important for the engineering of cultivars with tolerance to nonoptimal temperatures. Our study investigated the proteomic responses of rice cell suspension cultures to sudden temperature changes. Cell cultures grown at 28°C were subjected to 3‐day exposure to 12 or 20°C for low‐temperature stress, and 36 or 44°C for high‐temperature stress. Quantitative label‐free shotgun proteomic analysis was performed on biological triplicates of each treatment. Over 1900 proteins were expressed in one or more temperature treatments, and, of these, more than 850 were found to be responsive to either of the temperature extremes. These temperature‐responsive proteins included more than 300 proteins which were uniquely expressed at either 12 or 44°C. Our study also identified 40 novel stress–response proteins and observed that switching between the classical and the alternative pathways of sucrose metabolism occurs in response to extremes of temperature.     

Effect of Crystal Orientation and Conduction Band Grading of Absorber on Efficiency of Cu(In,Ga)Se2 Solar Cells Grown on Flexible Polyimide Foil at Low Temperature

Flexible Cu(In,Ga)Se₂ (CIGS) solar cells are developed on polyimide (PI) foil by using a three‐stage co‐evaporation method. To deposit CIGS on a PI substrate, low temperature deposition process (below 440 °C) is investigated. By optimizing the three‐stage process, power conversion efficiency (PCE) values of 12.1% and 13.6% are obtained at maximum process temperatures of 400 °C and 440 °C, respectively (without anti‐reflection coating). The CIGS absorber deposited at 400 °C is grown with (220)/(204) rather than (112) as its dominant growth orientation, accompanied by highly homogeneous crystal structure and phases. Admittance spectroscopy reveals that the defect levels were lower in the CIGS absorber with (220)/(204) dominant growth orientation, which could have contributed to the PCE of 12.1%. The CIGS solar cell deposited at a temperature of 440 °C exhibits worse defect characteristics compared to that fabricated at 400 °C. However, the CIGS solar cell fabricated below 440 °C exhibited marginal double‐grading of Ga/(Ga + In) in the absorber layer, which resulted in an improved PCE of 13.6%. This result implies that at a temperature below 440 °C, the double‐grading of Ga/(Ga + In) is the more dominant factor causing the PCE improvement above 13%.