Acquired thermotolerance and heat shock proteins in thermophiles from the three phylogenetic domains.

Thermophilic organisms from each of the three phylogenetic domains (Bacteria, Archaea, and Eucarya) acquired thermotolerance after heat shock. Bacillus caldolyticus grown at 60 degrees C and heat shocked at 69 degrees C for 10 min showed thermotolerance at 74 degrees C, Sulfolobus shibatae grown at 70 degrees C and heat shocked at 88 degrees C for 60 min showed thermotolerance at 95 degrees C, and Thermomyces lanuginosus grown at 50 degrees C and heat shocked at 55 degrees C for 60 min showed thermotolerance at 58 degrees C. Determinations of protein synthesis during heat shock revealed differences in the dominant heat shock proteins for each species. For B. caldolyticus, a 70-kDa protein dominated while for S. shibatae, a 55-kDa protein dominated and for T. lanuginosus, 31- to 33-kDa proteins dominated. Reagents that disrupted normal protein synthesis during heat shock prevented the enhanced thermotolerance.     

Inhibition of repair of X-ray-induced DNA damage by heat: the role of hyperthermic inhibition of DNA polymerase alpha activity

HeLa S3 cells growing in suspension have been used to investigate possible mechanisms underlying the inhibitory action of hyperthermia (44 degrees C) on the repair of DNA strand breaks as caused by a 6-Gy X-irradiation treatment. The role of hyperthermic inactivation of DNA polymerase alpha was investigated using the specific DNA polymerase alpha inhibitor, aphidicolin. It was found that both heat and aphidicolin (greater than or equal to 2 micrograms ml-1) could decrease DNA repair rates in a dose-dependent way. When the applications of heat and aphidicolin were combined, each at nonmaximal doses, no full additivity in effects was observed on DNA repair rates. When the heat and radiation treatment were separated in time by postheat incubation at 37 degrees C, restoration to normal repair kinetics was observed within 8 h after hyperthermia. When heat was combined with aphidicolin addition, restoration of the aphidicolin effect to control level was also observed about 8 h after hyperthermia. It is suggested that although DNA polymerase alpha seems to be involved in the repair of X-ray-induced DNA damage, and although this enzyme is partially inactivated by heat, other forms of heat damage have to be taken into account to explain the observed repair inhibition.     

Inhibition by hyperthermia of repair synthesis and chromatin reassembly of ultraviolet-induced damage to DNA

We have investigated the effects of hyperthermia treatment on sequential steps of the repair of UV-induced DNA damage in HeLa cells. DNA repair synthesis was inhibited by 40% after 15 min of hyperthermia treatment at 45 degrees C; greater inhibition of repair synthesis occurred with prolonged incubation at 45 degrees C. Enzymatic digestion of repair-labeled DNA with Exonuclease III indicated that once DNA repair was initiated, the DNA repair patch was synthesized to completion and that ligation of the DNA repair patch occurred. Thus the observed inhibition of UV-induced DNA repair synthesis by hyperthermia treatment may be the result of inhibition of enzymes involved in the initiating step(s) of DNA repair. DNA repair patches synthesized in UV-irradiated cells labeled at 37 degrees C with [3H]Thd were 2.2-fold more sensitive to micrococcal nuclease digestion than was parental DNA; if the length of the labeling period was prolonged, the nuclease sensitivity of the repair patch synthesized approached that of the parental DNA. DNA repair patches synthesized at 45 degrees C, however, remained sensitive to micrococcal nuclease digestion even after long labeling periods, indicating that heat treatment inhibits the reassembly of the DNA repair patch into nucleosomal structures.     

Induction of heat shock protein and thermal sensitivity of mammalian cells maintained at low culture temperature.

Effects of low culture temperature on the induction of heat shock proteins in FM3A cells by a heat shock and on the thermal sensitivity of the cells were examined. FM3A cells maintained at 33 degrees C could not induce hsp70 during continuous heating or after a short heat shock at either 39, 42, or 45 degrees C, although FM3A cells maintained at a normal culture temperature of 37 degrees C can induce the synthesis of hsp70. Furthermore, the cells maintained at 33 degrees C were more sensitive to the subsequent heat shock than the cells maintained at 37 degrees C. Thus, the culture temperature of the mammalian cells may be an important factor for the induction of hsp70, and hsp70 may play an important role to protect or repair the thermal damage of cells.     

Decline in mutation frequency in temperature-sensitive SV40 viruses before viral DNA synthesis.

Lesions that promote reversion from a temperature-sensitive to a wild-type phenotype were induced in temperature-sensitive late mutants of SV40 virus by UV irradiation. When cultures infected with UV-irradiated temperature-sensitive mutants were grown for various times at permissive temperature (35 degrees C) and then at restrictive temperature (39 degrees C), the reversion frequency declined just before the onset of semiconservative DNA synthesis when DNA synthesis began at 32 degrees C. This can be explained by competition between reactions that lead to the onset of viral DNA synthesis and reactions that repair the lesions before the onset of viral DNA synthesis.     

Heat shock protein 70 stimulation of the deoxyribonucleic acid base excision repair enzyme polymerase beta

Base excision repair (BER) of damaged deoxyribonucleic acid (DNA) is a multistep process during which potentially lethal abasic sites temporarily exist. Repair of these lesions is greatly stimulated by heat shock protein 70 (Hsp70), which enhances strand incision and removal of the abasic sites by human apurinic-apyrimidinic endonuclease (HAP1). The resulting single-strand gaps must then be filled in. Here, we show that Hsp70 and its 48- and 43-kDa N-terminal domains greatly stimulated filling in the single-strand gaps by DNA polymerase beta, a novel finding that extends the role of Hsps in DNA repair. Incorporation of deoxyguanosine monophosphate (dGMP) to fill in single-strand gaps in DNA phagemid pBKS by DNA polymerase beta was stimulated by Hsp70. Truncated proteins derived from the C-terminus of Hsp70 as well as unrelated proteins were less effective, but proteins derived from the N-terminus of Hsp70 remained efficient stimulators of DNA polymerase beta repair of DNA single-strand gaps. In agreement with these results, repair of a gap in a 30-bp oligonucleotide by polymerase beta also was strongly stimulated by Hsp70 although not by a truncated protein from the C-terminus of Hsp70. Sealing of the repaired site in the oligonucleotide by human DNA ligase 1 was not specifically stimulated by Hsp-related proteins. Results presented here now implicate and extend the role of Hsp70 as a partner in the enzymatic repair of damaged DNA. The participation of Hsp70 jointly with base excision enzymes improves repair efficiency by mechanisms that are not yet understood.     

Inviability of dam recA and dam recB cells of Escherichia coli is correlated with their inability to repair DNA double-strand breaks produced by mismatch repair.

The molecular basis for the inviability of dam-3 recA200(Ts) and dam-3 recB270(Ts) cells was studied. The dam-3 recA200(Ts) cells were inviable in yeast extract-nutrient broth or in minimal medium at 42 degrees C. Although the dam-3 recB270(Ts) cells were inviable in yeast extract-nutrient broth at 42 degrees C, they were viable at 42 degrees C in minimal medium, in which the high salt content suppresses the mutant phenotype caused by the recB270(Ts) mutation at 42 degrees C. Under the growth conditions rendering dam rec cells inviable, the cells accumulated double-strand breaks in their DNA. Introduction of a mutL or mutS mutation restored the viability of dam-3 recB270(Ts) cells grown in yeast extract-nutrient broth at 42 degrees C and eliminated the formation of DNA double-strand breaks in these cells. We conclude that the inability to repair DNA double-strand breaks produced by the mismatch repair process accounts for the inviability of the dam recA and dam recB cells.     

The cytoplasmic chaperone hsp104 is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum

Severe heat stress causes protein denaturation in various cellular compartments. If Saccharomyces cerevisiae cells grown at 24 degrees C are preconditioned at 37 degrees C, proteins denatured by subsequent exposure to 48-50 degrees C can be renatured when the cells are allowed to recover at 24 degrees C. Conformational repair of vital proteins is essential for survival, because gene expression is transiently blocked after the thermal insult. Refolding of cytoplasmic proteins requires the Hsp104 chaperone, and refolding of lumenal endoplasmic reticulum (ER) proteins requires the Hsp70 homologue Lhs1p. We show here that conformational repair of heat-damaged glycoproteins in the ER of living yeast cells required functional Hsp104. A heterologous enzyme and a number of natural yeast proteins, previously translocated and folded in the ER and thereafter denatured by severe heat stress, failed to be refolded to active and secretion-competent structures in the absence of Hsp104 or when an ATP-binding site of Hsp104 was mutated. During recovery at 24 degrees C, the misfolded proteins persisted in the ER, although the secretory apparatus was fully functional. Hsp104 appears to control conformational repair of heat-damaged proteins even beyond the ER membrane.     

Thermal inactivation of Enterococcus faecium: effect of growth temperature and physiological state of microbial cells

AIMS: To provide data on the effects on culture temperature and physiological state of cells on heat resistance of Enterococcus faecium, which may be useful in establishing pasteurization procedures. METHODS AND RESULTS: The heat resistance of this Ent. faecium (ATCC 49624 strain) grown at different temperatures was monitored at various stages of growth. In all cases, the bacterial cells in the logarithmic phase of growth were more heat sensitive. For cells which had entered in the stationary phase, D70 values of 0.53 min at 5 degrees C, 0.74 min at 10 degrees C, 0.83 min at 20 degrees C, 0.79 min at 30 degrees C, 0.63 min at 37 degrees C, 0.48 min at 40 degrees C and 0.41 min at 45 degrees C were found. By extending the incubation times cells were more heat resistant as stationary phase progressed, although a different pattern was observed for cells grown at different temperatures. At the lower temperatures heat resistance increased progressively, reaching D70 values of 1.73 min for cells incubated at 5 degrees C for 50 days and 1.04 min for those grown at 10 degrees C for 16 days. At other temperatures assayed heat resistance became stable for late stationary phase cells, reaching D70 values of 1.05, 1.08 and 1.01 min for cultures incubated at 20, 30 and 37 degrees C. Heat resistance of cells obtained at higher temperatures, 40 and 45 degrees C, was significantly lower, with D70 values of 0.76 and 0.67 min, respectively. Neither the growth temperature nor the growth phase modified the z-values significantly. CONCLUSIONS: D70 values obtained for Ent. faecium (ATCC 49624) varies from 0.33 to 1.73 min as a function of culture temperature and physiological state of cells. However, z values calculated were not significantly influenced by these factors. A mean value of 4.50 +/- 0.39 degrees C was found. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall results strongly suggest that, to establish heat processing conditions of pasteurized foods ensuring elimination of Ent. faecium, it is advisable to take into account the complex interaction of growth temperature and growth phase of cells acting on bacterial thermal resistance.     

Effects of several factors on the heat-shock-induced thermotolerance of Listeria monocytogenes.

The influence of the temperature at which Listeria monocytogenes had been grown (4 or 37 degrees C) on the response to heat shocks of different durations at different temperatures was investigated. For cells grown at 4 degrees C, the effect of storage, prior to and after heat shock, on the induced thermotolerance was also studied. Death kinetics of heat-shocked cells is also discussed. For L. monocytogenes grown at 37 degrees C, the greatest response to heat shock was a fourfold increase in thermotolerance. For L. monocytogenes grown at 4 degrees C, the greatest response to heat shock was a sevenfold increase in thermotolerance. The only survival curves of cells to have shoulders were those for cells that had been heat shocked. A 3% concentration of sodium chloride added to the recovery medium made these shoulders disappear and decreased decimal reduction times. The percentage of cells for which thermotolerance increased after a heat shock was smaller the milder the heat shock and the longer the prior storage.     

Leishmania amazonensis: effects of heat shock on ecto-ATPase activity

In this work we demonstrated that promastigotes of Leishmania amazonensis exhibit an Mg-dependent ecto-ATPase activity, which is stimulated by heat shock. The Mg-dependent ATPase activity of cells grown at 22 and 28 degrees C was 41.0+/-5.2 nmol Pi/h x 10(7)cells and 184.2+/-21.0 nmol Pi/h x 10(7)cells, respectively. When both promastigotes were pre-incubated at 37 degrees C for 2h, the ATPase activity of cells grown at 22 degrees C was increased to 136.4+/-10.6 nmol Pi/h x 10(7) whereas that the ATPase activity of cells grown at 28 degrees C was not modified by the heat shock (189.8+/-10.3 nmol Pi/h x 10(7)cells). It was observed that Km of the enzyme from cells grown at 22 degrees C (Km=980.2+/-88.6 microM) was the same to the enzyme from cells grown at 28 degrees C (Km=901.4+/-91.9 microM). In addition, DIDS (4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid) and suramin, two inhibitors of ecto-ATPases, also inhibited similarly the ATPase activities from promastigotes grown at 22 and 28 degrees C. We also observed that cells grown at 22 degrees C exhibit the same ecto-phosphatase and ecto 3'- and 5'-nucleotidase activities than cells grown at 28 degrees C. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat-shock effect on ecto-ATPase activity of cells grown at 22 degrees C were exposed at 37 degrees C for 2h. A comparison between the stimulation of the Mg-dependent ecto-ATPase activity of virulent and avirulent promastigotes by the heat shock showed that avirulent promastigotes had a higher stimulation than virulent promastigotes after heat stress.     

Heat protectors and heat-induced preferential redistribution of 26 and 70 kDa proteins in Chinese hamster ovary cells

An overall increase of 40% in nuclear-associated protein has been shown to be one of the sequellae of exposure of eukaryotic cells to elevated temperatures. Several investigators have shown that the increased protein/DNA ratios correlated well with the degree of cytotoxicity. In previous investigations, we have shown that cycloheximide, which protects the cell from the killing effects of heat, produces a dramatic reduction of the bulk nuclear-associated proteins after heating. In this investigation, we studied a previously unobserved efflux of a 26 kDa protein after heat shock and the preferential accumulation of the 70 kDa protein. The 26 kDa protein was shown not to be a member of previously described heat shock protein families. Preferential reduction of a 26 kDa protein and accumulation of a 70 kDa protein was observed in nuclei isolated from Chinese hamster ovary cells after heating at 43 degrees C. After heat treatment, the 26 kDa protein in the nucleus was decreased to a level 0.1-0.3 times the original amount in unheated cells, and the 70 kDa protein in the nucleus increased by a factor of 1.6-1.8. The normal levels of these two proteins were restored when cells were incubated at 37 degrees C following heat shock. Cells treated with heat protectors, cycloheximide and histidinol, demonstrated approximately the same redistribution in nuclear 26 and 70 kDa proteins immediately after heating as those not exposed to these drugs. On the other hand, restoration to control levels was much faster in the protector-treated cells, suggesting that "repair" of heat-induced damage is an important factor in the cells ability to survive this insult. Return to normal protein levels did not require new protein synthesis.     

A recombinant exonuclease III homologue of the thermophilic archaeon Methanothermobacter thermautotrophicus

AP endonucleases catalyse an important step in the base excision repair (BER) pathway by incising the phosphodiester backbone of damaged DNA immediately 5' to an abasic site. Here, we report the cloning and expression of the 774 bp Mth0212 gene from the thermophilic archaeon Methanothermobacter thermautotrophicus, which codes for a putative AP endonuclease. The 30.3 kDa protein shares 30% sequence identity with exonuclease III (ExoIII) of Escherichia coli and 40% sequence identity with the human AP endonuclease Ape1. The gene was amplified from a culture sample and cloned into an expression vector. Using an E. coli host, the thermophilic protein could be produced and purified. Characterization of the enzymatic activity revealed strong binding and Mg2+-dependent nicking activity on undamaged double-stranded (ds) DNA at low ionic strength, even at temperatures below the optimum growth temperature of M. thermautotrophicus (65 degrees C). Additionally, a much faster nicking activity on AP site containing DNA was demonstrated. Unspecific incision of undamaged ds DNA was nearly inhibited at KCl concentration of approximately 0.5 M, whereas incision at AP sites was still complete at such salt concentrations. Nicked DNA was further degraded at temperatures above 50 degrees C, probably by an exonucleolytic activity of the enzyme, which was also found on recessed 3' ends of linearized ds DNA. The enzyme was active at temperatures up to 70 degrees C and, using circular dichroism spectroscopy, shown to denature at temperatures approaching 80 degrees C. Considering the high intracellular potassium ion concentration in M. thermautotrophicus, our results suggest that the characterized thermophilic enzyme acts as an AP endonuclease in vivo with similar activities as Ape1.     

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.     

Effects of gamma radiation and hyperthermia on DNA repair synthesis and the level of NAD+ in cultured human mononuclear leukocytes

DNA repair has been investigated, estimated by unscheduled DNA synthesis (UDS) and the cellular NAD+ pool, after exposing human mononuclear leukocytes to hyperthermia and gamma radiation separately and in combination. It was found that gamma radiation induced a decline in UDS with increasing temperature through the temperature region studied (37-45 degrees C). At 42.5 degrees C the gamma-ray-induced UDS was reduced to about 70% of that at 37 degrees C. Following gamma-ray damage the NAD+ pool dropped to about 20% of control values. Without hyperthermic treatment the cells completely recovered to the original level within 5 hr. Moderate hyperthermia (42.5 degrees C for 45 min) followed by gamma-ray damage altered the kinetics so that even after 8 hr the NAD+ pool had recovered to only 70% of the original level. After heat treatment at 44 degrees C for 45 min prior to gamma radiation the cells did not recover at all, presumably because of the cytotoxic effects from the combined treatment.     

Acclimation of the photosynthetic machinery to high temperature in Chlamydomonas reinhardtii requires synthesis de novo of proteins encoded by the nuclear and chloroplast genomes

The mechanism responsible for the enhancement of the thermal stability of the oxygen-evolving machinery of photosystem II during acclimation of Chlamydomonas reinhardtii to high temperatures such as 35 degrees C remains unknown. When cells that had been grown at 20 degrees C were transferred to 35 degrees C, the thermal stability of the oxygen-evolving machinery increased and within 8 h it was equivalent to that in cells grown initially at 35 degrees C. Such enhancement of thermal stability was prevented by cycloheximide and by lincomycin, suggesting that the synthesis de novo of proteins encoded by both the nuclear and the chloroplast genome was required for this process. No increase in thermal stability was observed when cells that had been grown at 35 degrees C were exposed to heat shock at 41 degrees C, optimum conditions for the induction of the synthesis of homologs of three heat shock proteins (Hsps), namely, Hsp60, Hsp70, and Hsp22. Moreover, no synthesis of these homologs of Hsps was induced at 35 degrees C. Thus it appears likely that Hsps are not involved in the enhancement of the thermal stability of the oxygen-evolving machinery.     

Nuclear proteins in Drosophila melanogaster cells after heat shock and their binding to homologous DNA

After 5 minutes heat shock at 37 degrees C Drosophila melanogaster Kc-cell nuclear proteins were extracted wit 0.4M NaCl and compared by SDS gel electrophoresis with extracts from cells grown at 25 degrees C. Two proteins (39 000 and 46 000) were only found in heat shock nuclei. Reconstitution with total Drosophila DNA or a DNA fragment from the heat inducible locus 87A/C covalently coupled to sepharose was performed. In the presence of calf thymus competitor DNA these proteins and also others of lower molecular weight showed preferential binding to the homologous DNA.     

Organ culture of mammalian testis. III. Inhibin secretion.

Mouse testes were cultured for 19--20 days at either 31 or 37 degrees C with a change of medium every 4 days. After treatment with charcoal and dextran T, the recovered testis media were incubated with rat anterior pituitary cells, and secretions of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were estimated by radioimmunoassay 3 days later. FSH release was significantly lowered when pituitary cells were grown with media of testes cultured 31 degrees C compared to cultures grown with fresh medium or with media of testes cultured at 37 degrees C for more than 4 days. LH secretion was normal in one experiment and reduced in the other with the media of testes cultured at 31 degrees C. Treatment of testicular media by heat or trypsin reduced the inhibiting activity. After 8 days at 37 degrees C, both germinal and Sertoli cells were damaged in the testis cultures, while at 31 degrees germinal cells alone were destroyed, Sertoli cells remained normal. These studies suggest that (1) a substance which responds to the definition of inhibition (protein--preferentially acting on FSH) is secreted in the medium of testis culture; (2) inhibin is produced by Sertoli cells; (3) inhibin is secreted only if the temperature is inferior to 37 degrees C.