Suppression of an exocellular proteinase synthesis inBacillus megaterium by increased temperature

During cultivation ofBacillus megaterium at 42 °C the amount of the exocellular protease produced by growing cells sharply decreases as compared with temperatures of 28 and 35 °C. Within the above range the growth rate and incorporation of amino acids increase with increasing temperature. The culture adapted to 42 °C does not produce more proteinase at this temperature than the non-adapted culture. The high temperature does not induce accumulation of the enzyme in the cells. Total protein excretion was slightly lower at 42 °C than at 28 and 35 °C.     

Contact-mediated changes in cycloheximide-induced agglutinability of BHK cells

Suspension cultures of BHK cells grow in MEM supplemented with 10% fetal calf serum at about 50% the rate of corresponding monolayer cultures. If the serum supplement is reduced to 2% no increase in cell number is observed. When 10% serum is used small spheroids comprising 3–4 cells form within a 24 h period, but in 2 % serum the cells remain single over the same period. The addition of cycloheximide to contact-inhibited monolayer cultures induces high levels of ConA agglutinability within 6 h, yet growing non-confluent cells are rendered only about half as agglutinable by the same treatment. Cycloheximide treatment of suspension cultures causes growing cells to become agglutinable, but non-growing cells, which do not form spheroids, remain non-agglutinable even after 24 h of treatment. This suggests that the pronounced effect of cycloheximide on the agglutinability of contact-inhibited cells in monolayer culture reflects their confluence rather than suspended growth, and that the turnover rate of surface molecules determining the agglutinability state of cells is enhanced as cell-to-cell contact increases.     

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.     

Arabidopsis: Sensitivity of growth to high temperature

Arabidopsis thaliana seedlings as measured by an electrolyte leakage assay, have been found to be extremely sensitive to high temperature stress as compared to a high temperature tolerant variety (Tracy) of soybean. Over 50% ion leakage occurred in Arabidopsis leaves during a 15-minute exposure to 50°C, indicating a heat killing time of less than 15 minutes. In contrast, the heat killing time for soybean at 50°C was over five times longer. When soybean or Arabidopsis seedlings in culture plates were exposed to 37°C for 2 hours and then returned to 23°C, they suffered no apparent short-term or long-term damage. Soybean seedlings given a 42°C, treatment for 2 hours also showed no damage. Arabidopsis seedlings after a 42°C treatment for 2 hours showed no apparent immediate damage, but 48 hours after return to 23°C severe damage symptoms were visible and after 96 hours all the seedlings were dead. Both soybean and Arabidopsis seedlings synthesize heat shock proteins (hsps) when exposed to 42°C for 2 hours. The hsps synthesized are of similar molecular weights, although the relative abundances of the different size classes are very different in the two plants. Even though hsps are produced in Arabidopsis seedlings after a 2 hour exposure to 42°C their presence is not sufficient for the seedlings to recover from the effects of rhe heat shock when returned to 23°C. Our results show that Arabidopsis has a heat sensitive genotype. This along with its other characteristics should make it a good model system in which to assay in transgenic plants, the functions of homologous and heterologous genes that might be candidates for determining heat tolerance in plants.     

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.     

Short‐term high temperature treatment reduces viability and inhibits respiration and DNA repair enzymes in Araucaria angustifolia cells

We evaluated the effect of global warming on Araucaria angustifolia (Bert.) O. Kuntze, a critically endangered native tree of Southern Brazil, by studying the effects of short‐term high temperature treatment on cell viability, respiration and DNA repair of embryogenic cells. Compared with control cells grown at 25°C, cell viability was reduced by 40% after incubation at 30 and 37°C for 24 and 6 h, respectively, while 2 h at 40 and 42°C killed 95% of the cells. Cell respiration was unaffected at 30–37°C, but dramatically reduced after 2 h at 42°C. The in vitro activity of enzymes of the base excision repair (BER) pathway was determined. Apurinic/apyrimidine endonuclease, measured in extracts from cells incubated for 2 h at 42°C, was completely inactivated while lower temperatures had no effect. The activities of three enzymes of the mitochondrial BER pathway were measured after 30‐min preincubation of isolated mitochondria at 25–40°C and one of them, uracil glycosylase, was completely inhibited at 40°C. We conclude that cell viability, respiration and DNA repair have different temperature sensitivities between 25 and 37°C, and that they are all very sensitive to 40 or 42°C. Thus, A. angustifolia will likely be vulnerable to the short‐term high temperature events associated with global warming.     

A Cell Line (HEW) from Embryos of Haddock (Melanogrammus aeglefinius) and Its Capacity to Tolerate Environmental Extremes

Cell lines can be useful experimental tools for studying marine fish, which are often difficult to routinely obtain and maintain in the laboratory. As few cell lines are available from coldwater marine fish, cultures were initiated from late gastrula embryos of haddock (Melanogrammus aeglefinus) in Leibovitz's L-15 with fetal bovine serum (FBS). From one culture, a cell line (HEW) emerged that has been grown for close to 100 population doublings, was heteroploid, and expressed telomerase activity, all of which suggest HEW is immortal. Growth occurred only if FBS was present and was optimal at 12 to 18°C. Usually most cells had an epithelial-like morphology, but under some conditions, cells drew up into round central bodies from which radiated cytoplasmic extensions with multiple branches. These neural-like cells appeared within a few hours of cultures being placed at 28°C or being switch to a simple salt solution (SSS). At 28°C, cells died within 24 h. In SSS, HEW cells survived as a monolayer for at least 7 days. The sensitivity of HEW cells to morphological change and their capacity to withstand starvation should make them useful for investigating cellular responses to environmental stresses.     

Subnuclear redistribution of DNA species in confluent and growing mammalian cells

About 20 to 25 percent of the nuclear DNA from cultured cells of the African green monkey, Cercopithecus aethiops, consists of a homogeneous, highly repetitive fraction designated C. aethiops component DNA. Use of in situ hybridization techniques reveals component at the centromeres of chromosomes from both diploid and heteroploid African green monkey kidney (AGMK) tissue culture cells. — Component DNA comprises 47 percent of the nucleolar DNA in actively growing primary AGMK cells, but only 31 percent of the nucleolar DNA in confluent cells which show density-dependent growth inhibition. Further, there is a pronounced shift of both main band and component DNA from euchromatin to heterochromatin when growing cells attain confluency. Thus, the relative subnuclear distributions of component and main band DNA's are different in growing and confluent cells. — In situ hybridization techniques indicate that component sequences aggregate in clumps in nuclei of growing cells and show a diffuse distribution in nuclei of confluent cells. This suggests that centromeric regions of the various chromosomes or groups of chromosomes aggregate and disaggregate reversibly as the culture changes from density-dependent growth inhibition to active cell division. — Hypotonic citrate treatment of primary AGMK cells causes nucleoli of confluent cells to disperse: this dispersion following citrate treatment was not seen in growing AGMK cells or in confluent or growing heteroploid cells. Similarly, this nucleolar dispersion was seen in confluent diploid mouse and human cells but not in growing diploid cells or in confluent or growing heteroploid cells.     

Differences between diploid and transformed human cells in the incorporation of 5-bromodeoxyuridine into DNA

In diploid human cells, the DNA precursor pool equilibration times for exogenous thymidine are about twice those for the thymidine analogue 5-bromodeoxyuridine (BUdR); in cells that were either transformed chemically or derived from malignant tumours, the pool equilibration times are the same for thymidine and 5-bromodeoxyuridine and are closer in value to the shorter (bromodeoxyuridine) times of the diploid cells. Thymidine, if present in the culture medium with BUdR, is incorporated into DNA preferentially in diploid cells (by 2 or 3 to 1). Discrimination against bromodeoxyuridine is evident within 2 h of incubation of the two precursors with diploid cells, but is not observed even after 24 h in any of the transformed cell lines tested. Experiments were performed to test the effect of inhibitors of the mammalian DNA polymerases alpha (N-ethylmaleimide) and beta (incubation of cells at 45 °C) upon the ability of cells to synthesise DNA and to incorporate thymidine preferentially when present with equimolar BUdR. In diploid cells, overall in vivo DNA synthesis is more sensitive to N-ethylmaleimide and more resistant to 45 °C treatment than is DNA synthesis in the transformed cell lines. N-Ethylmaleimide decreases the capacity of diploid cells to discriminate against BUdR, whereas heating increases it. Transformed cells treated with N-ethylmaleimide remain unable to discriminate against BUdR; some transformed lines, when heated at 45 °C, become less incapable of such discrimination.     

Temperature-limited Activity in the Garter Snake Thamnophis melanogaster (Colubridae)

Many garter snakes, Thamnophis melanogaster, at a desert pond first started foraging for tadpoles when mean water surface temperature was about 20 °C (at 0945–1015 h), and the number of snakes tripled when water temperature reached about 24 °C (at 1100–1130 h). In two years, snakes foraged in April and May, but not in March when water never reached 23 °C and only exceeded 20 °C for a few hours after the usual foraging hours. Snakes in the laboratory dedicated increasing amounts of time to underwater foraging as air and water temperatures increased from 9 °C to 29 °C, and their rate of attacks on fish increased steeply and progressively above an apparent threshold lying between roughly 19 °C and 24 °C, up to at least 29 °C. Temperature may limit T. melanogaster's foraging at the pond to the hours after roughly 0900 h and to the period after roughly March, despite evidence that prey abundance is maximal in March.     

Increase in total protein following infection of CV-1 cells with SV40 virus as assayed by flow cytometry

Summary The changes in cell size and total protein were determined for G1-arrested, contact-inhibited CV-1 cells infected with Simian virus 40 (SV40). The assays used were the Biorad total protein assays (Bradford and DC protein assays) on a standard number of cells, total protein as assayed by fluorescein isothiocyanate (FITC) and SR101 by flow cytometry, orthoganol (90°) light scatter by flow cytometry, and direct microscopic measurement with an ocular micrometer. Uninfected CV-1 cells and two cell lines with variations in DNA content (diploid vs. tetraploid) were used as controls for the studies presented. The results demonstrated a 40–60% increase in total protein at 32 to 42 h postinfection. These increases were similar to values obtained as control cells progress through the cell cycle. At later times postinfection (>42 h), total protein decreased due to cellular changes resulting from viral replication and cell death.     

Regulation of protein synthesis in mammalian cells. V. Further studies on the effect of actinomycin D on translation control in HeLa cells

The rate of protein synthesis in HeLa cells appears to be regulated, in part, by a factor which promotes the association of ribosomes with messenger RNA and whose production is inhibited by actinomycin. The decline in protein synthesis after the administration of actinomycin is not primarily due to a decay of available messenger RNA but, rather, is a result of a decrease in the rate of ribosomal association with message.The decay of protein synthesis in actinomycin can be varied over a wide range by altering the temperature of cell incubation. Thus the half-life of protein synthesis decay ranges from eight hours at 34 °C to two hours at 41°C. The rapid decline of protein synthesis at 41 °C is not accompanied by a corresponding decay of the messenger RNA. Polyribosomes decrease in size, but they can be restored to normal sedimentation distributions by low levels of cycloheximide, suggesting that messenger RNA remains functional. The translation rate at 41 °C is unaltered. The dose-response of protein synthesis inhibition by actinomycin was measured and a half-maximum inhibition was found to be effected by 0·1 μg of the drug/ml.Another important aspect of the regulation of translation in HeLa cells is the response of cells to depressed rates of protein synthesis. At 42 °C, protein synthesis is severely inhibited, due to a failure in the association of ribosomes with messenger RNA. Prolonged incubation at the elevated temperature results in a significant repair of the lesion. This repair is inhibited by actinomycin. The half-maximum inhibition is achieved at levels of from 0·05 to 0·1 μg of the drug/ml.The cell response to depressed rates of protein synthesis can also be demonstrated using the drug cycloheximide. Prolonged incubation in the drug results in a response which then can promote protein synthesis at 42 °C. Here again, the half-maximum inhibition of the response to cyclohemixide is achieved by 0·1 μg of actinomycin/ml. These experiments suggest, but do not prove, that the cellular response may be mediated through the synthesis of RNA that promotes the initiation of translation and does not involve the subsequent production of protein.     

Kinetics of herpes simplex virus photoinhibition by haematoporphyrin in both diploid and heteroploid cells

The kinetics of inhibition of herpes simplex virus type 1 (HSV 1) on both diploid (CEF) and heteroploid cells (HEp2) by light-irradiated haematoporphyrin (HP) was studied. The inactivation of HSV1 by HP was drug-dose dependent and light-irradiation dependent; the viruses grown in heteroploid cells being in all cases more sensitive to inhibition than viruses grown in diploid cells. Cell toxicity by HP was markedly more evident on HEp2 cells than on CEF. The highest viral sensitivity to photodynamic inactivation by HP was found to be between the 4th and the 5th hour after cell infection, when the viral DNA synthesis is at its peak and before it is incorporated into complete virions. Microfluorometric and spectrofluorometric assays revealed that virus infected cells always take up more HP than uninfected cells, and heteroploid cells incorporated more HP than diploid cells. The possibility that an increased uptake of HP and modifications of the cell micro-environment in virus infected cells could account for the viral-inhibiting properties of HP, is discussed.     

Ploidy evolution in the yeast Saccharomyces cerevisiae: a test of the nutrient limitation hypothesis

The nutrient limitation hypothesis provides a nongenetic explanation for the evolution of life cycles that retain both haploid and diploid phases: differences in nutrient requirements and uptake allow haploids to override the potential genetic advantages provided by diploidy under certain nutrient limiting conditions. The relative fitness of an isogenic series of haploid, diploid and tetraploid yeast cells (Saccharomyces cerevisiae), which were also equivalent at the mating type locus, was measured. Fitness was measured both by growth rate against a common competitor and by intrinsic growth rate in isolated cultures, under four environmental conditions: (1) rich medium (YPD) at the preferred growth temperature (30 °C); (2) nutrient poor medium (MM) at 30 °C; (3) YPD at a nonpreferred temperature (37 °C); and (4) MM at 37 °C. In contrast to the predictions of the nutrient limitation hypothesis, haploids grew significantly faster than diploids under nutrient rich conditions, but there were no apparent differences between them when fitness was determined by relative competitive ability. In addition, temperature affected the relative growth of haploids and diploids, with haploids growing proportionately faster at higher temperatures. Tetraploids performed very poorly under all conditions compared. Cell geometric parameters were not consistent predictors of fitness under the conditions measured.     

Effect of Variation of Sporulation Time and Temperature on Thermostability of Bacillus cereus Spores

The optimum temperature for sporulation of a strain of Bacillus cereus was estimated at 30°–35°C, where the maximum yield of spores was obtained between 18 and 24 hours’ incubation. Sporulation was more rapid, but less extensive at 40°C and did not occur at all at 45°C. The heat resistance of the spores increased with the sporulation temperature from 20° to 40°C. The spores appear to be more susceptible to heat destruction in the early stage of spore production than after further incubation.     

Thermally Enhanced Radiosensitivity of Cultured Chinese Hamster Cells

X-IRRADIATION of mammalian cells in culture yields a survival curve of the threshold type (for review see ref. 1). It isjnter-esting to ask how one can enhance the radiation response by small changes of the physical environment of the cells, as can be done chemically, for example, by incorporation of 5-bromo-deoxyuridine into DNA^1,2. Elevation of the temperature is a likely prospect for enhancement of radiosensitivity for the following reasons. It is known that proteins are heat labile and that temperature sensitive mutants of bacteria and phage can be obtained for many different enzymes³ which are operative at 37° C but not at 42° or 43°C. For example⁴, DNA polymerase is reversibly temperature sensitive; it is rendered inoperative above 42°C, but will be functional again when the temperature is lowered. It is not unreasonable to expect that temperature sensitive mutations for many enzymes occur frequently and that the use of temperatures somewhat higher than the normal range at which the cells grow might disclose sensitivities for specific enzymes in normal cells of higher organisms.     

Growth and Dormancy in Norway Spruce Ecotypes (Picea abies) I. Interaction of Photoperiod and Temperature

Growth and dormancy as affected by photoperiod and temperature have been studied in Norway spruce ecotypes of different latitudinal and altitudinal origin. First-year seedlings were used. In all ecotypes apical growth cessation and terminal bud formation occurred within 2 weeks after exposure to SD at temperatures of 18 to 24°C. At lower temperatures or at near-critical photoperiods the response was delayed. The critical photoperiod for apical growth cessation varied from 21 hours in ecotype Steinkjer, Norway (64°N) to about 15 hours in ecotype Lankowitz, Austria (47°04′N). High-elevation ecotypes also had longer critical pholoperiods than low-elevation ecotypes from the same latitude. A detectable growth depression resulted from as little as 1 or 2 SDs of 10 hours, and with 4 or more SDs apical growth cessation took place. In contrast to the situation in the shoot, root growth was not affected by photoperiod. Accordingly, the top:root ratio is drastically affected by photoperiod. The critical photoperiod for cambial growth was shorter than that for apical growth in all ecotypes and cambial growth cessation was delayed for several weeks compared with cessation of apical growth. A transition to formation of late-wood tracheids with thick walls and narrow lumens took place upon exposure to SD. The photoperiodic effects were significantly modified by temperature, but the critical photoperiods were only slightly changed by temperature in the range of 12 to 24°C. However, a 10-hour “night” at 4°C caused growth cessation in continuous light in four ecotypes tested. Temperature optimum for apical growth under non-limiting photoperiods (24 hours) was 21°C in all ecotypes, but with little difference among 18,21 and 24°C. The Q₁₀ for apical growth was 3.5 in the temperature range 12 to 18°C. The growth potential as determined in 24-hour photoperiods was not significantly different among the various ecotypes except for one northern eco-type which was clearly inferior to the others. However, the growth of ecotype Steinkjer (64°N) was greatly suppressed even by the long midsummer days at 59°40′N, thus demonstrating the misleading impression one gets of the growth potential of northern ecotypes when they are moved southwards.     

Difference between diploid and aneuploid Chinese hamster cells in replication at mid-S-phase

Following partial synchronization of the heteroploid Chinese hamster cell line V-79 and of normal diploid lung fibroblasts of the Chinese hamster in culture, their DNA replication during S-phase was compared by means of a BrdU-incorporation/thymidine pulse technique and Hoechst-Giemsa differential staining of metaphase chromosomes. This comparison indirectly shows the S-phase of the heteroploid cells of V-79 to be 2 h shorter than the diploid cell S-phase. When the thymidine pulse is applied to diploid lung fibroblasts at mid-S-phase, differential staining colours metaphase chromosomes a pale blue. Performing the corresponding experiment with V-79 cells, neither a pale blue nor dark red staining is obtained, but rather an intermediate shade, showing prominently dark staining regions in parts. The pause in DNA synthesis observed at mid-S-phase of the diploid Chinese hamster lung fibroblasts seems to be omitted at mid-S-phase of the V-79 cells.     

The initiation and elongation steps in protein synthesis: Relative rates in chinese hamster ovary cells during and after hyperthermic and hypothermic shocks

The relative rates of the initiation and elongation phases of protein synthesis have been determined in heat- and cold-shocked CHO cells from measurements of the incorporation of ³⁵S-methionine into N-terminal and internal positions of growing peptides by a modified Edman degradation. When the cells are shifted from 37°C to temperatures between 10°C and 34°C, the rate of initiation is at first reduced more extensively than that of elongation. After 20 to 30 minutes at the lower temperature, however, the cells undergo a metabolic adjustment which includes increasing the rate of initiation until it corresponds to the rate of elongation at that temperature. Calculated apparent energies of activation for initiation and elongation are in reasonable agreement with those determined in other mammalian cells. When the cooled cells are returned to 37°C, the rates of initiation and elongation recover immediately but do not exceed the control values. Exposure to elevated temperature (43°C) causes an immediate cessation of initiation and thus a delayed inhibition of elongation; upon return to 37°C, the rate of initiation is transiently elevated above the control rate, and the rate of elongation returns to the control rate after a 2- to 3-minute delay. Hence, a factor which leads to supranormal rates of initiation may accumulate at high but not at low temperatures.