Role of glutathione and hsp 70 in the acquisition of thermotolerance in postimplantation rat embryos

Studies were initiated to determine the extent to which reduced glutathione (GSH) may be involved in the capacity of cultured rat embryos to develop heat-induced tolerance to the deleterious effects of exposure to high temperatures (heat shock). Investigations of the modulation of dysmorphogenic responses of embryos to heat shock (43 degrees C, 30 min) as well as to the expression of the hsp70 gene and subsequent formation of hsps indicated that the acquisition of thermotolerance by rat embryos could be significantly influenced by the inhibition of GSH synthesis. Treatment of conceptuses with L-buthionine-S,R-sulfoximine (BSO) reduced intracellular GSH concentrations and compromised the capacity of embryos to mount a thermotolerance response as assessed by alterations in indices of growth and development. Embryonic thermotolerance elicited by preexposure to 42 degrees C for 30 min was accompanied by increases in GSH to levels greater than those measured in control embryos at 37 degrees C just prior to the subsequent 43 degrees C heat exposure. Expression of hsp70 mRNA was detectable soon after elevation of the temperature to 42 degrees C and reached its highest level of accumulation 1.5 hr after the 43 degrees C heat shock. BSO treatment had little if any effect on hsp70 message levels or on the synthesis of hsp70. The fact that BSO-treatment attenuated the thermotolerance response but did not produce a decrease in hsp70 RNA or the synthesis of hsp70 suggests that hsp70 alone is not sufficient to confer thermotolerance upon cultured rat embryos.     

Hyperthermia-induced heat shock response and thermotolerance in postimplantation rat embryos

Postimplantation stage rat embryos (6-10 somites) undergo abnormal development after exposure to a temperature of 43 degrees C for 30 min. A heat shock of 43 degrees C for 30 min also induces the synthesis of a set of eight heat shock proteins (hsps) with molecular masses ranging from 28,000 to 82,000 Da. The synthesis of these hsps is rapidly induced after the heat shock is applied and rapidly decays after embryos are returned to 37 degrees C. A heat shock of 42 degrees C for 30 min has no effect on rat embryo growth and development, but does induce the synthesis of three hsps. The most prominent of these three is believed to be the typical mammalian 70 kDa hsp. Furthermore, a 42 degrees C, 30-min heat shock followed by a 43 degrees C 30-min heat shock leads to partial protection from the embryotoxic effects of a single exposure at 43 degrees C, i.e., thermotolerance.     

Loss of heat-shock acquisition of thermotolerance in yeast is not correlated with loss of heat-shock proteins

Yeast cells when subjected to a primary heat shock, defined as a temperature shift from 23 to 37 degrees C for 30 min, acquired tolerance to heat stress (52 degrees C/5 min). Primary heat shocked cells incubated at 23 degrees C for up to 3 h, progressively lost thermotolerance but retained high levels of the major heat-shock proteins as observed on polyacrylamide gels. On the other hand, a temperature shift back up to 37 degrees C for 30 min fully restored thermotolerance. The major high-molecular-mass heat-shock proteins (hsp) identified were of approximate molecular mass 100 kDa (hsp 100), 80 kDa (hsp 80) and 70 kDa (hsp 70). The results indicate that loss of heat-shock acquisition of thermotolerance is not correlated with loss of heat-shock proteins.     

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 cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.     

Induced thermotolerance in bovine two-cell embryos and the role of heat shock protein 70 in embryonic development

Induced thermotolerance is a phenomenon whereby exposure to a mild heat shock can induce heat shock proteins (HSP) and other cellular changes to make cells more resistant to a subsequent, more severe heat shock. Given that the 2-cell bovine embryo is very sensitive to heat shock, but can also produce HSP70 in response to elevated temperature, experiments were conducted to test whether 2-cell embryos could be made to undergo induced thermotolerance. Another objective was to test the role of the heat-inducible form of heat shock protein 70 (HSP70i) in development and sensitivity of bovine embryos to heat shock. To test for induced thermotolerance, 2-cell bovine embryos were first exposed to a mild heat shock (40 degrees C for 1 hr, or 41 degrees C or 42 degrees C for 80 min), allowed to recover at 38.5 degrees C and 5% (v/v) CO2 for 2 hr, and then exposed to a severe heat shock (41 degrees C for 4.5, 6, or 12 hr). Regardless of the conditions, previous exposure to mild heat shock did not reduce the deleterious effect of heat shock on development of embryos to the blastocyst stage. The role of HSP70i in embryonic development was tested in two experiments by culturing embryos with a monoclonal antibody to the inducible form of HSP70. At both 38.5 degrees C and 41 degrees C, the proportion of 2-cell embryos that developed to blastocyst was reduced (P < 0.05) by addition of anti-HSP70i to the culture medium. In contrast, sensitivity to heat shock was not generally increased by addition of antibody. In conclusion, bovine 2-cell embryos appear incapable of induced thermotolerance. Lack of capacity for induced thermotolerance could explain in part the increased sensitivity of 2-cell embryos to heat shock as compared to embryos at later stages of development. Results also implicate a role for HSP70i in normal development of bovine embryos.     

Acquisition of the heat-shock response and thermotolerance during early development of Xenopus laevis

The ability to synthesize a 68,000- to 70,000-Da protein (hsp) in heat-shocked early Xenopus laevis embryos is dependent on the stage of development. Whereas late blastula and later stage embryos synthesize hsp68-70 after heat shock, cleavage stages are incompetent with respect to hsp synthesis. In vitro translation experiments and RNA blot analyses demonstrate that enhanced synthesis of hsp68-70 is associated with an accumulation of hsp68-70 mRNA. Examination of the effect of heat shock on preexisting actin mRNA reveals that heat shock promotes a reduction in the levels of actin mRNA in cleavage embryos but has no discernible effect on actin mRNA levels in neurula embryos. Finally, the acquisition of the heat-shock response (i.e., synthesis of hsp68-70 and accumulation of hsp70 mRNA) during early Xenopus development is correlated with the acquisition of thermotolerance.     

Heat shock-induced acquisition of thermotolerance at the levels of cell survival and translation in Xenopus A6 kidney epithelial cells.

In this study we have investigated the acquisition of thermotolerance in a Xenopus laevis kidney A6 epithelial cell line at both the level of cell survival and translation. In cell survival studies, A6 cells were incubated at temperatures ranging from 22 to 35 degrees degrees C for 2 h followed by a thermal challenge at 39 degrees degrees C for 2 h and a recovery period at 22 degrees C for 24 h. Optimal acquisition of thermotolerance occurred at 33 degrees degrees C. For example, exposure of A6 cells to 39 degrees degrees C for 2 h resulted in only 3.4% survival of the cells whereas prior exposure to 33 degrees C for 2 h enhanced the survival rate to 69%. This state of thermotolerance in A6 cells was detectable after 1 h at 33 degrees C and was maintained even after 18 h of incubation. Cycloheximide inhibited the acquisition of thermotolerance at 33 degrees C suggesting the requirement for ongoing protein synthesis. The optimal temperature for the acquisition of translational thermotolerance also occurred at 33 degrees C. Treatment of A6 cells at 39 degrees C for 2 h resulted in an inhibition of labeled amino acid incorporation into protein which recovered to approximately 14% of control after 19 h at 22 degrees C whereas cells treated at 33 degrees C for 2 h prior to the thermal challenge recovered to 58% of control levels. These translationally thermotolerant cells displayed relatively high levels of the heat shock proteins hsp30, hsp70, and hsp90 compared to pretreatment at 22, 28, 30, or 35 degrees C. These studies demonstrate that Xenopus A6 cells can acquire a state of thermotolerance and that it is correlated with the synthesis of heat shock proteins.     

The 70-kilodalton heat-shock proteins of the SSA subfamily negatively modulate heat-shock-induced accumulation of trehalose and promote recovery from heat stress in the yeast, Saccharomyces cerevisiae.

In the yeast, Saccharomyces cerevisiae, the disaccharide trehalose is a stress-related metabolite that accumulates upon exposure of cells to heat shock or a variety of non-heat inducers of the stress response. Here, we describe the influence of mutations in individual heat-shock-protein genes on trehalose metabolism. A strain mutated in three proteins of the SSA subfamily of 70-kDa heat-shock proteins (hsp70) overproduced trehalose during heat shock at 37 degrees C or 40 degrees C and showed abnormally slow degradation of trehalose upon temperature decrease from 40 degrees C to 27 degrees C. The mutant cells were unimpaired in the induction of thermotolerance; however, the decay of thermotolerance during recovery at 27 degrees C was abnormally slow. Since both a high content of trehalose and induced thermotolerance are associated with the heat-stressed state of cells, the abnormally slow decline of trehalose levels and thermotolerance in the mutant cells indicated a defect in recovery from the heat-stressed state. A similar albeit minor defect, as judged from measurements of trehalose degradation during recovery, was detected in a delta hsp104 mutant, but not in a strain deleted in the polyubiquitin gene, UB14. In all our experiments, trehalose levels were closely correlated with thermotolerance, suggesting a thermoprotective function of trehalose. In contrast, heat-shock proteins, in particular hsp70, appear to be involved in recovery from the heat-stressed state rather than in the acquisition of thermotolerance. Cells partially depleted of hsp70 displayed an abnormally low activity of neutral trehalase when shifted to 27 degrees C after heat shock at 40 degrees C. Trehalase activity is known to be under positive control by cAMP-dependent protein kinases, suggesting that hsp70 directly or indirectly stimulate these protein-kinase activities. Alternatively, hsp70 may physically interact with neutral trehalase, thereby protecting the enzyme from thermal denaturation.     

Stress proteins and stress tolerance in an Antarctic, psychrophilic yeast, Candida psychrophila

Conditions are described for the heat shock acquisition of thermotolerance, peroxide tolerance and synthesis of heat shock proteins (hsps) in the Antarctic, psychrophilic yeast Candida psychrophila. Cells grown at 15°C and heat shocked at 25°C (3 h) acquired tolerance to heat (35°C) and hydrogen peroxide (100 mM). Novel heat shock inducible proteins at 80 and 110 kDa were observed as well as the presence of hsp 90, 70 and 60. The latter hsps were not significantly heat shock inducible. The absence of hsp 104 was intriguing and it was speculated that the 110 kDa protein may play a role in stress tolerance in psychrophilic yeasts, similar to that of hsp 104 in mesophilic species.     

Heat shock response of Saccharomyces cerevisiae mutants altered in cyclic AMP-dependent protein phosphorylation.

When Saccharomyces cerevisiae cells grown at 23 degrees C were transferred to 36 degrees C, they initiated synthesis of heat shock proteins, acquired thermotolerance to a lethal heat treatment given after the temperature shift, and arrested their growth transiently at the G1 phase of the cell division cycle. The bcy1 mutant which resulted in production of cyclic AMP (cAMP)-independent protein kinase did not synthesize the three heat shock proteins hsp72A, hsp72B, and hsp41 after the temperature shift. The bcy1 cells failed to acquire thermotolerance to the lethal heat treatment and were not arrested at the G1 phase after the temperature shift. In contrast, the cyr1-2 mutant, which produced a low level of cAMP, constitutively produced three heat shock proteins and four other proteins without the temperature shift and was resistant to the lethal heat treatment. The results suggest that a decrease in the level of cAMP-dependent protein phosphorylation results in the heat shock response, including elevated synthesis of three heat shock proteins, acquisition of thermotolerance, and transient arrest of the cell cycle.     

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.     

Regulation of heat shock protein 70 synthesis by heat shock in the preimplantation murine embryo

Induced thermotolerance in murine embryos occurs at the 8-cell stage when embryos are maintained in vitro but not until the blastocyst stage if development proceeds in vivo. Present results indicate that ability of embryos to undergo induced thermotolerance is not limited by heat shock protein 70 (HSP70) synthesis. Exposure of 8-cell embryos to 40 degrees C enhanced synthesis of 2 constitutive HSP70 proteins (HSC70 and HSC72) and induced another protein, HSP68; exposure of 43 degrees C was required to induce similar responses in expanded blastocysts. Unlike induced thermotolerance, increased synthesis of HSP70 molecules did not depend on whether embryos were cultured or developed in vivo. Thus, other biochemical mechanisms in addition to HSP70 confer thermotolerance in the preimplantation-stage murine embryo. The observation that the temperature threshold for induction of HSP70 synthesis increased from the 8-cell to the blastocyst stage is indicative of these other biochemical processes.     

Patterns of protein synthesis in various cells after extreme heat shock

The analysis of proteins synthesized in rat thymocytes and mouse teratocarcinoma PCC-4 Aza 1 and myeloma Sp2/0 cells after 1 h of treatment at 42 or 44 degrees C was carried out. Shock at 42 degrees C reduced the total synthetic rate of proteins in all three cell lines and induced "classical" heat-shock protein with a mass of 70 kDa (hsp 70). Heat shock at 44 degrees C resulted in almost complete inhibition of protein synthesis; only a small amount of hsp 70 was synthesized. Meanwhile a new 48-kDa polypeptide (pI = 7.5) was found in the cells exposed to severe heat shock. This protein was compared by peptide mapping with other known polypeptides of the same size: heat-shock protein from chicken embryo cells and mitogen-stimulated polypeptide from human lymphoid cells. The peptide maps were not identical. It was also shown that after a shock at 44 degrees C teratocarcinoma cells were able to accumulate anomalous amounts of hsp 70 despite hsp 70 synthesis inhibition. The data show that reaction of various cells to extreme heat shock depends heavily on cell type.     

Stress-induced, tissue-specific enrichment of hsp70 mRNA accumulation in Xenopus laevis embryos

In this study, we have employed whole-mount, in situ hybridization to study the spatial pattern of hsc70 and hsp70 mRNA accumulation in normal and heat shocked embryos during Xenopus laevis development. Our findings revealed that hsc70 mRNA was constitutively present in a global fashion throughout the embryo and was not heat inducible. Accumulation of hsp70 mRNA, however, was detected only in heat shocked embryos. Furthermore, hsp70 mRNA accumulation was enriched in a tissue-specific manner in X. laevis tailbud embryos within 15 minutes of a 33 degrees C heat shock. Abundant levels of heat shock-induced hsp70 mRNA were detected in the head region, including the lens placode, the cement gland, and in the somitic region and proctodeum. Preferential heat-induced accumulation of hsp70 mRNA was first detected at a heat shock temperature of 30 degrees C. Placement of embryos at 22 degrees C after a 1-hour, 33 degrees C heat shock resulted in decreased hsp70 mRNA with time, but the message persisted in selected tissues, including the lens placode and somites. Treatment of tailbud embryos with either sodium arsenite or zinc chloride induced a tissue-specific enrichment of hsp70 mRNA in the lens placode and somitic region. These studies reveal the complex nature of the heat shock response in different embryonic tissues and suggest the presence of regulatory mechanisms that lead to a stressor-induced, tissue-specific enrichment of hsp70 mRNA.     

Relationship between cell survival and heat-stress protein synthesis in a Drosophila cell line

Heat-stress protein (hsp) kinetics and clonogenic survival were studied at 33, 37 and 42 degrees C in a continuous Drosophila cell line, WR69-DM-1. Induction and repression of hsp were temperature-dependent and independently modulated. The subsequent cell-survival curves were complex; however, survival generally decreased in a time- and temperature-dependent manner during continuous heating at 33, 37 or 42 degrees C. Constant 33 degrees C heating induced five hsp at 90, 72, 70, 24 and 19 kilodaltons (kDa). A 30 min 33 degrees C heat dose led to thermotolerance after 1, 3 or 6 h incubations at 28 degrees C. The hsp synthesized after this dose were quickly repressed, suggesting the cells were able to respond to this stress. Increasing the challenge temperature to 37 degrees C induced three additional hsp at 34, 22 and 14 kDa, but hsp synthesis did not lead to thermotolerance over the 6 h interval. The number and intensity of hsp synthesized was higher and repression was much slower than at 33 degrees C. Heating at 42 degrees C inhibited all protein synthesis, and thermotolerance was not observed. Direct survival data are critical to understanding the role and function of hsp in Drosophila thermotolerance since the relevance of information on number and kinetics of hsp synthesis and their subsequent localization is dubious without it.     

Acquisition of thermotolerance in Saccharomyces cerevisiae without heat shock protein hsp 104 and in the absence of protein synthesis.

Acquisition of thermotolerance in response to a preconditioning heat treatment at 40 degrees C was studied in mutants of the yeast Saccharomyces cerevisiae lacking a specific heat shock protein or the ability to synthesize proteins at 40 degrees C. A mutant carrying a deletion of heat shock protein hsp 104 and the corresponding wildtype strain were both highly sensitive to heat stress at 50.4 degrees C without preconditioning but both acquired almost the same level of thermotolerance after 60 min of preconditioning. Both strains showed equal induction of trehalose-6-phosphate synthase and accumulated equal levels of trehalose during the treatment. The conditional mutant ts--187 synthesized no proteins during the preconditioning heat treatment but nevertheless acquired thermotolerance, albeit to a lesser degree than the corresponding wildtype strain. Induction of trehalose-6-phosphate synthase and accumulation of trehalose were reduced to a similar extent. These results show that acquisition of thermotolerance and accumulation of trehalose are closely correlated during heat preconditioning and are modulated by protein synthesis but do not require it.     

Expression of heat shock protein in broiler embryo tissues after acute cold or heat stress

This study evaluated the expression of heat shock protein 70 kD (hsp70) in broiler chicken embryos subjected to cold (Experiment I) or high incubation temperature (Experiment II). In each experiment, fertile eggs were distributed in three incubators kept at 37.8 degrees C. At day 13 (D13), D16, and D19 of incubation, the embryos were subjected to acute cold (32 degrees C) or heat (40 degrees C) for 4-6 hr. Immediately after cold or heat exposure, samples from the liver, heart, breast muscle, brain, and lungs of 40 embryos were taken per age and treatment (control or stressed embryos). A tissue pool from 10 embryos was used as 1 replication. The levels of hsp70 in each tissue sample was quantified by Western blot analysis. The data were analyzed in a 3 x 2 factorial arrangement of treatments with four replications. hsp70 was detected in all embryo tissues, and the brain contained 2- to 5-times more hsp70 protein compared to the other tissues in either cold or heat stressed embryos. hsp70 increases were observed in the heart and breast muscle of cold stressed embryos at D16 and D19, respectively. Heat stressed embryos showed an increase of hsp70 in the heart at D13 and D19, and in the lung at D19 of incubation. Younger embryos had higher hsp70 synthesis than older embryos, irrespective of the type of thermal stressor. The results indicate that the expression of hsp70 in broiler chicken embryos is affected by cold and heat distress, and is tissue- and age-dependent.     

Time course and magnitude of synthesis of heat-shock proteins in congeneric marine snails (Genus tegula) from different tidal heights

The time course and magnitude of the heat-shock response in relation to severity of thermal stress are important, yet poorly understood, aspects of thermotolerance. We examined patterns of protein synthesis in congeneric marine snails (genus Tegula) that occur at different heights along the subtidal to intertidal gradient after a thermal exposure (30 degrees C for 2.5 h, followed by 50 h recovery at 13 degrees C) that induced the heat-shock response. We monitored the kinetics and magnitudes of protein synthesis by quantifying incorporation of 35S-labeled methionine and cysteine into newly synthesized proteins and observed synthesis of putative heat-shock proteins (hsp's) of size classes 90, 77, 70, and 38 kDa. In the low- to mid-intertidal species, Tegula funebralis, whose body temperature frequently exceeds 30 degrees C during emersion, synthesis of hsp's commenced immediately after heat stress, reached maximal levels 1-3 h into recovery, and returned to prestress levels by 6 h, except for hsp90 (14 h). In contrast, in the low-intertidal to subtidal species, Tegula brunnea, for which 2.5 h at 30 degrees C represents a near lethal heat stress, synthesis of hsp's commenced 2-14 h after heat stress; reached maximal levels after 15-30 h, which exceeded magnitudes of synthesis in T. funebralis; and returned to prestress levels in the case of hsp90 (50 h) and hsp77 (30 h) but not in the case of hsp70 and hsp38. Exposures to 30 degrees C under aerial (emersion) and aquatic (immersion) conditions resulted in differences in hsp synthesis in T. brunnea but not in T. funebralis. The different time courses and magnitudes of hsp synthesis in these congeners suggest that the vertical limits of their distributions may be set in part by thermal stress.