Effect of temperature on insulin binding and action in cultured human fibroblasts

Insulin stimulation of glycogen synthase activity and insulin binding were measured in fibroblast monolayers at 24, 32, and 37°C. Insulin stimulation of %$\text{I}$ glycogen activity increased with increasing temperature. Maximum response was greater at 37°C than at 32°C, and half maximal stimulation required at 2.0 nM insulin at 37°C vs. 10nM at 32°C. Insulin stimulation of glycogen synthase was greater and somewhat faster at 37°C than at 32°C. No insulin effect was observed at 24°C. ¹²⁵I-insulin binding to monolayers became maximal in 15 min at 37°C, 60 min at 32°C, and 120 min at 24°C. However, insulin binding decreased with increasing temperature, and this decline was due to decreased numbers of receptors. Insulin binding and stimulation of glycogen synthase were comparable at 32°C, with half maxima at 10 nM, indicating no evidence of “spare” receptors. The data indicate that temperature effects on insulin binding and action in fibroblasts are not directly related. The results also suggest that a rate limiting step(s) of insulin action is temperature sensitive, and that this step is not insulin binding.     

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.     

Phospholipid metabolism in Mycobacterium smegmatis ATCC 607 grown at 37° C and 27° C

The rate of synthesis and degradation of phospholipids in Mycobacterium smegmatis ATCC 607, grown at 27° C and 37° C was studied by incorporation of ³²P into phospholipids and chase of radioactivity of the pulse-labelled phospholipids. A relatively low rate of synthesis and degradation of phospholipids in cells growth at 27° C was observed as compared to those grown at 37° C. Phosphatidylethanolamine (PE) had the maximum turnover at 37° C. However, at 27° C, cardiolipin (CL) showed a turnover rate higher than PE. Phosphatidylinositol mannosides (PIMs) were metabolically more active at 37° C than at 27° C. The differences in metabolic activity of the phospholipids at the two temperatures have been discussed.     

Enhancement of productivity of recombinant α-amidating enzyme by low temperature culture

We have produced a recombinant C-terminal α-amidating enzyme (799BglIIα-AE) derived from Xenopus laevis by culturing a CHO cell line named 3μ-1S. Recently, we demonstrated that culturing 3μ-1S cells at a temperature below 37 °C led to the following phenomena: inhibited cell growth with high viability, enhanced cellular productivity (maximally at 32 °C), and suppressed medium consumption and release of impurities from the cells. Therefore, it is suggested that the 799BglIIα-AE production will be increased by culturing a sufficient number of the cells at a low temperature (especially at 32 °C). To assess this effect on batch and perfusion cultures, the culture temperature was shifted from 37 to 32 °C in the mid-exponential phase in the case of batch culture and from 37 to 34 °C when the cell density became high enough in the case of perfusion culture. Application of the low temperature culture to batch and perfusion cultures was effective in comparison with the culture at 37 °C: the productivity per medium and the productivity per time were increased severalfold with enhanced cellular productivity at a low culture temperature. The low temperature culture also increased the relative content of 799BglIIα-AE in the supernatant and reduced the glucose consumption. The method presented here would contribute to production of bioactive proteins using other recombinant cell lines. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effect of incubation temperature on the maintenance of rat palatal mucosa in organ culture

Summary The effect of incubation temperature on the behavior of neonatal rat palatal mucosa maintained in a chemically defined medium in organ culture for periods up to 7 days was investigated. Explant survival was optimal at 37°C with increasing mortality at temperatures of 34°C and 30°C. There was a transient increase in the epithelial mitotic activity at all temperatures, but at all time intervals mitotic activity was greatest at 37°C. While the mitotic activity at 37°C after 5 hr in vitro was comparable with previously described in vivo values, it was subsequently increased, only returning to values approximating those at the start of the experiment at 6 days. At 30° and 34° C the epithelial mitotic activity increased more slowly than at 37° C; then it followed a similar pattern with time and after 5 days in vitro had fallen to values approximating initial values. At the cut edges of the explants, the rate of epithelial migration and subsequent keratinization increased with increasing temperature. It is suggested that survival of neonatal rat palatal mucosa is optimal in this organ culture system when maintained at 37° C. This work forms part of a thesis submitted to the University of London for the degree of Doctor of Philosophy by M. W. H.     

Effect of temperature regimes on gibberellin levels in thermosensitive dwarf apple trees

Orchard-grown dwarf apple (Malus domestica Borkh.) trees selected from a hybrid population were propagated by tissue culture but had a growth pattern similar to standard cv. Golden Delicious plants when grown at constant 27°C instead of the expected dwarf pattern of growth. Shoot elongation was markedly reduced, with or without gibberellin A₁ (GA₁) or GA₄ treatment, when trees were grown in an environment where day temperature was maintained at 35°C for 2 h in a ramped regime (night 20°C day ramped to 35°C, held for 2 h and ramped down to 20°C night over a 14-h photoperiod). Application of GA₁ or GA₄ partially overcame growth retardation resulting from prior paclobutrazol treatment of both standard and dwarf trees grown at constant 27°C and of standard trees grown in the ramped environment. However, these GAs had no effect on paclobutrazol-treated or untreated dwarfs grown in the ramped regime. Gas chromatography-mass spectrometry with labelled internal standards was used to quantify GA₁, GA₃, GA₈, GA₁₉, GA₂₀ and GA₂₉ in extracts from standard and dwarf plants grown either at a constant 27°C or in a 20-30-20°C ramped temperature regime. Standard plants, which elongate quite rapidly in either environment, had similar levels of these GAs in both temperature regimes. The slowly growing dwarfs in the ramped temperature environment contained three times more GA₁₉ than the rapidly elongating dwarfs grown at 27°C. The concentrations of the other GAs were reduced to ca 40% or less in plants grown in the ramped temperature regime compared with those grown at 27°C. These data suggest that shoot elongation of dwarf plants is sensitive to elevated temperatures both as a result of reduced responsiveness to GAs and because of a reduction in the concentration of GA₁, apparently as a result of a lower rate of conversion of GA₁₉ to GA₂₀. It is possible that the altered GA metabolism may be a consequence of the change in GA sensitivity.     

Surfacing activity and food utilization in a tropical air-breathing fish exposed to different temperatures

Reared in tubular aquaria containing different depths of water (2.5, 5.0, 15.5, 31.0 and 40.0 cm), the obligatory air-breathing fish Ophiocephalus striatus (760 mg; 4.5 cm L) was forced to swim vertically a longer or shorter distance per surfacing. Interaction of temperature (17, 22, 27, 32 and 37°C) and aquarium depth reveals that surfacing frequency of the fish, fed ad libitum on Tilapia muscle, increased with increasing aquarium depth, but the increase was significant only at 27 and 32°C; in the starving series, the frequency was not depth-dependent at any temperature. Owing to the sustained surfacing activity and the consequent fatigue, the test individuals ‘hung’ to the surface for a definite period. Hanging frequency was temperature-dependent, but not a depth-dependent activity either in the starving or feeding series. At any temperature and aquarium depth, the feeding series hung more frequently than the starving series. Hanging duration increased from about 1 hr/day in either series at 17°C to 6 and 18 hr/day in the feeding and starving series at 37°C. At any tested temperature, distance swum by the feeding and starving series was a depth-dependent activity. The feeding series at 32°C exhibited the maximum swimming speed of 2 L/sec for 4.8 hr/day in the 40 cm depth. With increasing temperature and depth, feeding rate increased (from 24 to 225 g cal/g live fish/day); between 17 and 27°C, it was more a temperature-dependent activity. The highest rate (47 g cal / g/day) and efficiency (27%) of conversion were observed at 32°C; whereas the efficiency was depth-dependent, the rate was not. Oxygen uptake was a temperature-dependent activity; aquarium depth played a secondary role. Briefly, O. striatus in deeper aquaria consumed significantly more food and converted lesser, as it surfaced more frequently and swam longer distance, dissipating more energy on metabolism and swimming activity. Hence, culturing O. striatus in shallow waters at the optimum temperature of 32°C will be advantageous. This work was supported by the University Grants Commission's (New Delhi) grant to Dr. T. J. Pandian: Grant No. F. 23-210/75 (Sr II) for which appreciation is expressed.     

EVOLUTIONARY ADAPTATION TO TEMPERATURE. VI. PHENOTYPIC ACCLIMATION AND ITS EVOLUTION IN ESCHERICHIA COLI

Acclimation refers to reversible, nongenetic changes in phenotype that are induced by specific environmental conditions. Acclimation is generally assumed to improve function in the environment that induces it (the beneficial acclimation hypothesis). In this study, we experimentally tested this assumption by measuring relative fitness of the bacterium Escherichia coli acclimated to different thermal environments. The beneficial acclimation hypothesis predicts that bacteria acclimated to the temperature of competition should have greater fitness than do bacteria acclimated to any other temperature. The benefit predicted by the hypothesis was found in only seven of 12 comparisons; in the other comparisons, either no statistically demonstrable benefit was observed or a detrimental effect of acclimation was demonstrated. For example, in a lineage evolutionarily adapted to 37°C, bacteria acclimated to 37°C have a higher fitness at 32°C than do bacteria acclimated to 32°C, a result exactly contrary to prediction; acclimation to 27°C or 40°C prior to competition at those temperatures confers no benefit over 37°C acclimated forms. Consequently, the beneficial acclimation hypothesis must be rejected as a general prediction of the inevitable result of phenotypic adjustments associated with new environments. However, the hypothesis is supported in many instances when the acclimation and competition temperatures coincide with the historical temperature at which the bacterial populations have evolved. For example, when the evolutionary temperature of the population was 37°C, bacteria acclimated to 37°C had superior fitness at 37°C to those acclimated to 32°C; similarly, bacteria evolutionarily adapted to 32°C had a higher fitness during competition at 32°C than they did when acclimated to 37°C. The more surprising results are that when the bacteria are acclimated to their historical evolutionary temperature, they are frequently competitively superior even at other temperatures. For example, bacteria that have evolved at either 20°C or 32°C and are acclimated to their respective evolutionary temperatures have a greater fitness at 37°C than when they are acclimated to 37°C. Thus, acclimation to evolutionary temperature may, as a correlated consequence, enhance performance not only in the evolutionary environment, but also in a variety of other thermal environments.     

Influence of soil temperature on niacin and thiamine in honey mesquite

Summary The influence of soil temperature was examined on niacin and thiamine concentration in honey mesquite (Prosopis glandulosa var.glandulosa) seedlings. The seedlings were grown in soil temperature regimes of 21, 27, and 32°C in a controlled environment growth room. Nodulation randomly occurred on the roots of the seedlings, necessitating separate analysis according to the occurrence of nodulation. Roots of nodulated seedlings from the 21°C soil temperature regime contained greater quantities of niacin and thiamine compared to root samples from seedlings grown in either 27 or 32°C regimes. Niacin concentration of non-nodulated seedlings was highest in samples from seedlings grown in the 27°C soil temperature regime and lowest in samples from seedlings grown in the 21°C regime. Thiamine concentration was the greatest from non-nodulated seedlings grown in the 27°C soil temperature regime, while the thiamine concentration of non-nodulated samples from the 32°C regime was the least. Optimal soil temperature for honey mesquite root growth appears to be about 27°C. At sub-optimal soil temperatures niacin might have limited ‘growth’ while at supra-optimal soil temperatures, thiamine might be a limiting factor. College of Agricultural Sciences Contribution No. T-9-164.     

Effect of temperature on the development, fecundity, progeny sex ratio and life-table of Campoletis chlorideae, an endolarval parasitoid of the pod borer, Helicoverpa armigera

Development, survival, fecundity, progeny sex ratio (PSR) and age-specific life-table parameters of the parasitoid Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae) were examined at six different constant temperatures (12, 17, 22, 27, 32 and 37°C) in the laboratory [70 ± 10% RH and 10:14 h (light:dark) photoperiod]. Second instar larvae of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) were reared on chickpea (Cicer arietinum L.) and used as the host. Development times shortened as the temperature increased from 12 to 37°C. The estimated lower developmental threshold (tL) was 3.4°C. The thermal summation for total immature stages was 379.97 degree-days. A reciprocal relationship between temperature and longevity was observed in the range of 12–17°C. The maximum mortality of pupae (71.8%) occurred at 37°C. At 22°C, the yield of a female parasitoid averaged 137.3 ± 14.7 (mean ± SD) progeny, of which 89.6 ± 7.6 were daughters. The number of daughters produced decreased when the females were kept either above or below 22°C, although the PSR was female biased in the range of 17–27°C. The analyses of life-table parameters, developmental rates, reproduction, mortality and PSR suggest that maximum population growth (r _m ) is near 27°C. There was little variation observed in most of the desired qualities of C. chlorideae in the range of 17–27°C, and it appears that the parasitoid is adapted to a wide range of temperatures. We suggest that for maximum production the parasitoid should be reared at 22 ± 4°C and be released in areas where the temperature ranges between 17° and 27°C, as in the plains of northern India.     

The promotive effect on subsequent germination of treating imbibed celery seeds with high temperature before or during drying

Abstract High temperature (32°C) prevented germination of celery seeds even if given after 4 d of germination induction at 17°C in white light, but germination occurred if the seeds were then returned to 17°C. Celery seeds incubated for 3 d at 17°C in white light and then air-dried at 20°C germinated slowly when re-sown at 17°C in the light, achieving only 24% germination after 21 d. Exposure of such seeds to 32°C prior to and during drying resulted in 50% germination after 3.6 d at 17°C in white light, with no loss in viability, compared to 5.7 d for seeds not given a germination induction treatment. If celery seeds were dried rapidly germination was poor, an effect which could be overcome by high temperature treatment. It is suggested that the mechanism which imposes dormancy at 32°C also conditions the seed to withstand desiccation damage.     

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.     

Conditional inactivation of Aspergillus nidulans sarASAR1 uncovers the morphogenetic potential of regulating endoplasmic reticulum (ER) exit

In the genetic model Aspergillus nidulans, hyphal growth is exquisitely dependent on exocytic traffic. Following mutagenic PCR and gene replacement, we characterized thermosensitive mutations in sarA^SAR1 encoding a key regulator of endoplasmic reticulum (ER) exit. Six sarA^ts alleles permitting relatively normal growth at 30°C prevented it at 42°C. This growth phenotype correlated with markedly reduced SarA levels at high temperature, suggesting that these alleles cause temperature‐dependent SarA misfolding. sarA8 results in Ser substitution for conserved P‐loop Gly27. sarA5 (Trp185Cys) and sarA6 (Ser186Pro) substitutions underscore the importance of the C‐terminal α‐helix on SarA^Sar1 function/stability. sarA6 markedly diminishing growth at 37°C was useful for microscopy experiments in which ER exit was impaired by shifting the incubation temperature. Early and late Golgi cisternae, labeled with the integral membrane syntaxins SedV^Sed5 and TlgB^Tlg2, respectively, were rapidly dissipated by sarA6. However, whereas SedV^Sed5 was shifted toward the ER, TlgB^Tlg2 relocalized to a haze, underscoring the asymmetry of Golgi organization. This rapid Golgi dissipation that takes place after blocking anterograde COPII traffic is consistent with the cisternal maturation model. Incubation of sarA6 cells at 37°C led to the formation of apical balloons resembling specialized fungal structures. The formation of these balloons highlights the morphogenetic consequences of impairing ER exit.     

EVOLUTIONARY ADAPTATION TO TEMPERATURE. I. FITNESS RESPONSES OF ESCHERICHIA COLI TO CHANGES IN ITS THERMAL ENVIRONMENT

We used bacteria to study experimentally the process of genetic adaptation to environmental temperature. Replicate lines of Escherichia coli, founded from a common ancestor, were propagated for 2,000 generations in 4 different thermal regimes as 4 experimental groups: constant 32, 37, or 42°C (thermal specialists), or a daily alternation between 32 and 42°C (32/42°C: thermal generalists). The ancestor had previously been propagated at 37°C for 2,000 generations. Adaptation of the groups to temperature was measured by improvement in fitness relative to the ancestor, as estimated by competition experiments. All four experimental groups showed improved relative fitness in their own thermal environment (direct response of fitness). However, rates of fitness improvement varied greatly among temperature groups. The 42°C group responded most rapidly and extensively, followed by the 32 and 32/42°C groups, whose fitness improvements were indistinguishable. The 37°C group, which experienced the ancestral temperature, had the slowest and least extensive fitness improvement. The correlated fitness responses of each group, again relative to the common ancestor, were measured over the entire experimental range of temperatures. No necessary tradeoff between direct and correlated responses of fitness was apparent: for example, the improved fitness of the 42°C group at 42°C was not accompanied by a loss of fitness at 37°C or 32°C. However, the direct fitness responses were usually greater than the correlated responses, judged both by comparing direct and correlated responses of a single group at different temperatures and by comparing direct and correlated responses of different groups at a single temperature. These comparisons indicate that the observed adaptation was, in fact, largely temperature specific. Also, the fitness responses of the generalist group across a range of temperatures were less variable than those of the thermal specialist groups considered as whole.     

Multiple generation effects of high temperature on the development and fecundity of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype B

Insects are ectotherms and their ability to resist temperature stress is limited. The immediate effects of sub‐lethal heat stress on insects are well documented, but longer‐term effects of such stresses are rarely reported. In this study, survival, development and reproduction of the whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype B, were compared over five consecutive generations at 27, 31 and 35 °C and for one generation at 37 °C. Both temperature and generation significantly affected the fitness of the whitefly. These impacts were more dramatic with increasing generations and temperatures. Among the experimental temperatures, the most favorable for development and reproduction were 27 °C and 31 °C. At 27 °C, survival, development and fecundity were all stable over these five generations. At 31 °C, immature survival rate was the highest in the fifth generation, but female fecundities decreased in the fourth and fifth generations. At 35 °C, egg hatching rate, immature survival rate and female fecundity decreased significantly in the fourth and fifth generations. At 37 °C, survival of B. tabaci was not adversely affected, but female fecundity at 37 °C was less than 10% of that at 27 °C or 31 °C. These results demonstrate that the lethal high temperature for B. tabaci is over 37 °C, and the whitefly population continued expanding in the five generations at 35 °C. The ability of B. tabaci biotype B to survive high temperature stress will play an important role in its population extension under global warming.     

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.     

Influence of low-temperature storage and glucose starvation on growth recovery in Escherichia coli relA and relA+ strains.

To study the influence of microgravity on bacterial growth behavior during a space mission, the special experimental conditions and the hardware environment necessitate storage of cells at low temperature, and permit a relatively short experimental period. Before this experimental period, cells have to recover their condition of steady-state growth, because it is only in this condition that the growth behavior of the flight and ground populations can be adequately compared. To meet these requirements and to obtain cells which recover rapidly their steady-state growth, we analyzed the size and shape of Escherichia coli cells during storage at 4 degrees C, with and without previous glucose starvation of the cells. It appeared that cells stored at low temperature in the presence of glucose continued to increase in average mass and assumed ovoid shapes. In addition, upon restoration of maximal growth rate at 37 degrees C, they continued to increase in size and showed a transient overshoot of their final steady-state value, which was reached after about 5 h. Cells previously starved for glucose, however, maintained their average size and rod-shape during low-temperature storage. Recovery of the starved cells was most rapid in the relA+ strain which, contrary to the isogenic relA strain, showed no overshoot and reached its final steady-state size within 2 h.     

Growth and temperature relationships for juvenile fish species in seagrass beds: implications of climate change

The effect of water temperature on growth responses of three common seagrass fish species that co‐occur as juveniles in the estuaries in Sydney (34° S) but have differing latitudinal ranges was measured: Pelates sexlineatus (subtropical to warm temperate: 27–35° S), Centropogon australis (primarily subtropical to warm temperate: 24–37° S) and Acanthaluteres spilomelanurus (warm to cool temperate: below 32° S). Replicate individuals of each species were acclimated over a 7 day period in one of three temperature treatments (control: 22° C, low: 18° C and high: 26° C) and their somatic growth was assessed within treatments over 10 days. Growth of all three species was affected by water temperature, with the highest growth of both northern species (P. sexlineatus and C. australis) at 22 and 26° C, whereas growth of the southern ranging species (A. spilomelanurus) was reduced at temperatures higher than 18° C, suggesting that predicted increase in estuarine water temperatures through climate change may change relative performance of seagrass fish assemblages.     

Glutamic Acid Production in Biotin-rich Media by Temperature-sensitive Mutants of Brevibacterium lactofermentum,a Novel Fermentation Process

Temperature-sensitive mutants were derived from Brevibacterium lactofermentum strain 2256 in a search for mutants which would produce a large amount of L-glutamic acid in biotin- rich media at the nonpermissive temperature. A total of 159 mutant strains was selected which showed adequate growth at 30°C but showed little or no growth at 37°C on minimal medium. Twenty of these were found to produce glutamic acid in a biotin-rich medium after a temperature shift from 30°C to 37°C, while the wild-type strain 2256 did not produce it under the same cultural condition.

One of the typical mutant strains, Ts-88, produced approximately 2g/dl of glutamic acid from beet molasses (the yield > 55%) in the presence of 33 µg/liter of biotin when tempera- , ture was shifted from 30°C to 40°C during the cultivation. It was concluded that, by controlling only temperature during fermentation, glutamic acid production could be realized in media containing biotin-rich natural carbon sources, without any chemical control such as the addition of expensive surface-active agents or antibiotics. Characteristics and merits of the novel fermentation process are discussed.     

Cytolytic T lymphocyte mediated chromium-51 release versus spontaneous release from blast and spleen cell targets at low temperatures

The rate of spontaneous ⁵¹Cr release from spleen cell and LPS blast target cells is strongly temperature dependent. Between 32 and 37 °C the rate of spontaneous release increases dramatically with temperature. Cytolytic T-lymphocyte-mediated lysis of these target cells is also temperature dependent, but lysis does not increase greatly above 32 °C. The ratio of specific ⁵¹Cr release to spontaneous release can be significantly improved by doing ⁵¹Cr-release assays at temperatures below 37 °C.