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.     

Heat-shock proteins in membrane vesicles of Bacillus subtilis

Fractionation of B. subtilis cells after heat shock, from 37 degrees C to 54 degrees C, shows an increase in synthesis of proteins localized in cell membranes and a decrease in synthesis of proteins localized in cytosol. There is no such effect of heat shock at temperature of 45 degrees C. Autoradiograms of electrophoretically separated proteins, labelled during heat shock at 54 degrees C, reveal 26 heat-shock proteins (hsps) in membrane vesicles and 11 hsps in cytosol, five of which are common to both fractions. Heat shock at 45 degrees C induces 18 hsps localized in membrane vesicles and 13 hsps localized in cytosol, six of which are common to both fractions. Results are interpreted as showing a relevant role of membrane proteins in cell response to shock at high temperature, pointing to two steps of defense against heat stress.     

Heat shock proteins and thermotolerance in a cultured cell line from the Mediterranean fruit fly, Ceratitis capitata.

Heat shock proteins (hsps) were identified in a cell line from the Mediterranean fruit fly, Ceratitis capitata Wiedemann (Diptera: Tephritidae) exposed to elevated temperatures. Cells produced three hsps (Mr 87,000, 69,000, and 34,000) in response to a temperature shift from 26 degrees C to 37 degrees C (30-60 min) with a concomitant decrease in synthesis of most other cellular proteins. Synthesis of low Mr hsps was not evident. The heat shock response is triggered within 30 min at temperatures from 33 degrees C to 41 degrees C. At temperatures greater than 41 degrees C protein synthesis was shut down. Within 2-3 h after return to 26 degrees C, synthesis of proteins repressed at the higher temperatures resumed production while the major hsps disappear. Heat shock proteins were not produced in the presence of actinomycin D. Evaluations on the role of hsps in conferring thermotolerance to the cells showed an increase in cell viability in heat-shocked cells over non-heat-shocked cells (after 3 and 10 days) when subsequently placed at 45 degrees C for 1 h, a normally lethal temperature. Heat shock alone had little effect on subsequent cell viability or growth at 26 degrees C. These results suggest that hsps produced by these cells may aid in the maintenance of cell integrity and thus play a transitory role in thermotolerance.     

Factors influencing the heat shock response of Xenopus laevis embryos

We have further characterized the heat shock response of Xenopus laevis embryos. Xenopus embryos respond to heat shock by consistently synthesizing four major heat shock proteins (hsps) of 62, 70, 76, and 87 kilodaltons. In addition to these hsps, heat-shocked embryos also exhibit the synthesis of several minor hsps. The synthesis of these hsps is often variable. We have monitored the effects of different temperatures and lengths of heat shock on the pattern and intensity of hsp synthesis. In general, the four major hsps are induced more strongly at higher temperatures and during increasing intervals of heat shock. The temperature and duration of heat shock can affect the synthesis of the minor hsps, however. Some hsps are synthesized at lower temperatures only (i.e., below 37 degrees C), whereas others are synthesized only at higher temperatures (i.e., above 37 degrees C). We have extensively examined the characteristics of hsp 35 synthesis, one of the most variably synthesized hsps. This hsp is characteristically synthesized at temperatures above 35 degrees C and usually during the first 40 min of heat shock, after which it becomes undetectable. In some experiments, its synthesis is restimulated during later intervals of heat shock. Hsp 35 is also under developmental regulation. It is not synthesized by heat-shocked embryos until the late blastula to early gastrula stage. After this brief period of inducibility, its synthesis is dramatically reduced in mid- to late gastrulae, but reappears in heat-shocked neurulae. We have previously demonstrated that hsp 35 is related to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The induction of hsp 35 synthesis is inversely correlated with the constitutive levels of GAPDH specific activity. In this paper we document further correlations between the synthesis of hsp 35 and GAPDH specific activity during early Xenopus development.     

Regulation of heat-shock protein synthesis in chicken muscle culture during recovery from heat shock

Exposure of chick myotube cultures to a temperature (45 degrees C) higher than their normal growing temperature (37 degrees C) caused extensive synthesis of three major polypeptides of Mr = 25 000, 65 000 and 81 000 referred to as 'heat-shock polypeptides' (hsps). When these cells were allowed to recover from heat-shock treatment at 37 degrees C for 6-8 h, the rate of accumulation of isotope into the 65 000-Mr and 81 000-Mr hsps declined to levels comparable to those in control cultures maintained at 37 degrees C. However, incorporation of isotope in the 25 000-Mr hsp continued at an elevated rate for a longer period than the 65 000-Mr and 81 000-Mr hsps. When heat-shocked cells were allowed to recover at 37 degrees C in the presence of actinomycin D to block new mRNA synthesis, the hsp synthesis as measured by the incorporation of radioactive isotope in these polypeptides continued at levels comparable to those in heat-shocked cells prior to recovery. The block of recovery by actinomycin D was due to the presence of a greater amount of functional hsp mRNAs in the polysomes as compared to untreated controls. The role of competition between the mRNAs for hsps and normal cellular proteins for the translation machinery in regulating protein synthesis during the recovery from heat shock has been discussed.     

Dissociation of 68,000 Mr heat shock protein synthesis from thermotolerance expression in rat fibroblasts

Rat embryonic fibroblasts growing exponentially at either 35, 37, or 39 degrees C were exposed to 42 degrees C for times up to 6 hr. Cell survival was unaffected by this heat shock in cultures growing at 39 degrees C but survival was decreased in a temperature dependent manner in cells growing at 37 or 35 degrees C. Exposure to 42 degrees C of cells previously adapted to 35 or 37 degrees C resulted in the induction of heat shock proteins (hsps) with apparent molecular weights of 68,000 (hsp 68), 70,000 (hsp 70), and 89,000 (hsp 89); cells previously adapted to 39 degrees C expressed all hsps except hsp 68. Inasmuch as the synthesis of certain hsps may function to protect cells from thermal damage, these data indicate that hsp 68 may not be required for this adaptation-related thermotolerant survival response. Hsp 68 may only be expressed in cells destined to die.     

Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves.

Biochemical and electron microscopic analyses of heat-shocked suspension cultures of Peruvian tomato (Lycopersicon peruvianum) revealed that a considerable part of the dominant small heat shock proteins (hsps) with an Mr of approximately 17,000 are structural proteins of newly forming granular aggregates in the cytoplasm (heat shock granules), whose formation strictly depends on heat shock conditions (37 to 40 degrees C) and the presence or simultaneous synthesis of hsps. However, under certain conditions, e.g., in preinduced cultures maintained at 25 degrees C, hsps also accumulate as soluble proteins without concomitant assembly of heat shock granules. Similar heat shock-induced cytoplasmic aggregates were also observed in other cell cultures and heat-shocked tomato leaves and corn coleoptiles.     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

Heat shock proteins do not provide thermoprotection to normal cellular protein synthesis, α-amylase mRNA and endoplasmic reticulum lamellae in barley aleurone layers

Heat shock in barley ( Hordeum vulgare L. cv. Himalaya) aleurone layers induces the synthesis of heat shock proteins (hsps) and suppresses the synthesis and secretion of α-amylase, the principal secretory protein. This is accompanied by the destabilization of α-amylase mRNA and a concomitant dissociation of ER lamellae. In the absence of heat shock α-amylase mRNA is extremely stable (Belanger et al. 1986. Proc. Natl. Acad. Sci. USA 83: 1354–1358). In most organisms there is a direct correlation between the synthesis of hsps and thermotolerance. The ability of hsps to provide thermoprotection to secretory protein synthesis, α-amylase mRNA and ER lamellae was analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of pulse-chased, [³⁵S]-methionine-labeled proteins revealed that the half-life of hsps in barley aleurone cells recovering from heat shock was approximately 12 h. Within approximately 6 h, there was a recovery of α-amylase mRNA and a reformation of ER lamellae. Heat shock protein synthesis was induced by either heat shock (40°C) or arsenite, the cells were allowed to recover for 8 h, then were re-exposed to heat shock. Results from SDS-PAGE showed that, despite the presence of hsps, α-amylase synthesis was suppressed. Northern blot hybridizations showed that α-amylase mRNA levels were reduced in heat-shocked tissues. Transmission electron microscopy demonstrated that ER lamellar structures were dissociated. The synthesis of hsps did not enable barley aleurone cells to sustain the synthesis of any proteins at lethal temperature. In contrast, similar conditions established thermotolerance and provided thermoprotection to protein synthesis in germinating barley embryos. Our findings suggest that the aleurone layer does not become thermotolerant following the induction of hsp synthesis.     

Stage dependent synthesis of heat shock induced proteins in early embryos of Drosophila melanogaster

Summary The synthesis of heat shock proteins (hsp) has been examined during the early embryogenesis of Drosophila melanogaster. Normal protein synthesis stops after heat shock at all developmental stages, while hsp synthesis is induced only after treatment at blastoderm and later stages. The small hsps continue to be synthesised after heat shock for a longer period than the larger ones. Heat shocks at 35°C, 37°C and 40°C were compared for their effect on hsp synthesis and the effect of heat shock on the normal course of development was analysed.     

A nuclear protein associated with lethal heat shock of HL-60 cells.

The responses to stress in living cells are well known. Thermal stress causes decreased protein synthesis as well as rapid induction of heat shock proteins (hsps), or alternately termed stress proteins (sps). The exposure of cultured promyelocytic leukemia cells (HL-60) to a 45 degrees C lethal heat shock for 1 h elicited synthesis and phosphorylation of a polypeptide M(r) 48,000 and pI 7.5 (p 48) as visualized by two-dimensional polyacrylamide gel ultra-microelectrophoresis. p 48, which was not observed at sublethal temperatures (39 and 41 degrees C), was synthesized during all phases of the cell cycle but was phosphorylated only in G0 + G1 and S-phases. The appearance of p 48 was marked by a concomitant and reciprocal reduction in hsps or sps 70 and 90. Distinct protease V8 fragment maps of p 48, hsps 70 and 90 in conjunction with immunochemical determination indicated vast differences in their primary structures. These facts suggest that p 48 was not formed from coalesced breakdown products of hsps 70 or 90. Western blotting showed that p 48 possessed the same immunochemical determinants as two other proteins with the same molecular mass but different isoelectric points. In an association assay, p 48 was shown to bind with actins and hsp 90 from HL-60 nuclei.     

Cryoprotection provided by heat shock treatment in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells exposed to 43 degrees C (normal being 30 degrees C) exhibit the synthesis of heat shock proteins (hsps). Time course studies indicated that the major hsps (97 kDa, 85 kDa and 70 kDa family) are induced within 10 min. of heat shock and attain maximum amount with two hours of treatment. The viability of cells decreased by 99% when directly frozen into liquid nitrogen. However, a prior heat shock (2 hours) increased the cell survival by 20-30 fold. Such an effect of prior heat shock treatment could be supported by light and electron microscopical studies. Differential scanning calorimetric analysis of whole cells revealed that heat shock treatment decreases the denaturation (delta H) of total cellular proteins. A direct correlation between the degree of hsp inducibility and protection against freezing and thawing injury was observed. Cycloheximide treatment curtailed the synthesis of hsps as well as protection against subsequent freezing. This suggests that prior heat shock treatment protects the cells from freezing injury and, furthermore, that hsps can act as biological cryoprotectants.     

The role of heat shock proteins in the osmoregulation of Escherichia coli

E. coli has a number of biochemical systems which protect cells from different chemical and physical damages. The aim of this work is to characterize the interaction between two of these: the osmoregulation system and the heat shock system. It is shown that exposure of E. coli to 42 degrees C to induce hsps synthesis, abolish the growth inhibition by high (0.45 M) NaCl concentration. Also, transient pretreatment of cells with high NaCl protect them from heat damage. It is shown that osmotic shock induces the hsps synthesis. The cell growth restoration after the complete inhibition by high (0.6 M) NaCl concentration correlates with the hsps accumulation. Moreover the heat shock treatment reduces the adaptation time.     

Heat shock proteins and chilling sensitivity of mung bean hypocotyls

Excised mung bean (Vigna radiata L.) hypocotyl sections wereexposed to 40 C for up to 4 h in the presence or absence of50 µM cycloheximide (CHX) before being held at a non-chilling(20 C) or chilling (2.5 C) temperature. Mung bean hypocotyltissue is chilling sensitive, and the rate of solute leakageis highly correlated with the extent of chilling injury. A 3h heat shock at 40 C reduced chilling-induced solute leakageby up to 40%, but leakage was similar to non-heat-shocked hypocotylswhen CHX was present. Specific proteins were labelled when hypocotylswere exposed to [³⁵S] methionine during the last hour of heatshock. The nine most intense bands on the autoradiographs ofSDS-PAGE gels of extracted protein corresponded to molecularweights of 114, 79, 73, 70, 60, 56, 51, 46, and 18 kDa. The18 kDa band reached a maximum after 1 h at 40 C and then rapidlydecreased in intensity as the heat shock continued, becomingundetectable at 4 h. The four most intense bands after 3 h at40 C corresponded to molecular weights of 79, 70, 51, and 46kDa. The synthesis of these four hsps was markedly reduced whenthe hypocotyl sections were exposed to CHX during heat shock.During chilling for 6 d, the levels of hsps 79 and 70 remainedsignificantly higher in tissue that was heat shocked prior tochilling than in tissue that was not heat shocked. In contrast,the levels of hsps 51 and 46 were similar in bothheat-shockedand control tissues. Heat-shock-induced chilling tolerance waslost between 6 and 9 d ofstorage at 2.5 C; this loss coincidedwith the decay of hsps 79 and 70 to control levels. These resultssuggest that heat shock induces an increase in both chillingtolerance and the de novo synthesis of specific heat shock proteins;namely hsps 79 and 70. This is the first report showing a relationshipbetween heat-shock-induced chilling tolerance and specific heat-shock-inducedproteins. Key words: Ion leakage, protein synthesis, Vigna radiata     

Heat shock protein induction and the acquisition of thermotolerance in the psychrotrophic yeastTrichosporon pullulans

Two-dimensional gel electrophoretic analysis of the heat shock response in the psychrotrophic yeastTrichosporon pullulans revealed the induction of 11 heat shock proteins (hsps) after a 5° to 21°C heat shock, 12 hsps after a 5° to 26°C heat shock, and 12 hsps after a 5° to 29°C heat shock. Heat shock from 5° to 26° or 29°C resulted in a statistically significant increase in thermotolerance to a lethal heat challenge at 45°C for 5 min. When the protein synthesis inhibitor, cycloheximide, was added prior to the heat shock, no statistically significant thermotolerance was acquired. To confirm the correlation between the synthesis of hsps and the acquisition of thermotolerance, protein extracts of cells that had been heat shocked in the presence or absence of cycloheximide were electrophoretically analyzed. Addition of the same concentration of cycloheximide that prevented the acquisition of thermotolerance also inhibited the synthesis of any hsps.     

Inhibition of heat shock protein synthesis and thermotolerance by cycloheximide

Chinese hamster ovary (CHO) cells were exposed to a 43 degrees C, 15-min heat shock to study the relationship between protein synthesis and the development of thermotolerance. The 43 degrees C heat shock triggered the synthesis of three protein families having molecular weights of 110,000, 90,000, and 65,000 (HSP). These proteins were synthesized at 37 and 46 degrees C. This heat shock also induced the development of thermotolerance, which was measured by incubating the cells at 46 degrees C 4 h after the 43 degrees C heat treatment. CHO cells were also exposed to 20 micrograms/ml of cycloheximide for 30 min at 37 degrees C, 15 min at 43 degrees C, and 4 h at 37 degrees C. This treatment inhibited the enhanced synthesis of the Mr 110,000, 90,000, and 65,000 proteins. The cycloheximide was then washed out and the cells were incubated at 46 degrees C. HSP synthesis did not recover during the 46 degrees C incubation. This cycloheximide treatment also partially inhibited the development of thermotolerance. These results suggest that for CHO cells to express thermotolerance when exposed to the supralethal temperature of 46 degrees C protein synthesis is necessary.     

The heat shock response inLocusta migratoria

Summary Locusta migratoria adults reared at 27–30°C die after 2 h at 50°C, but they survive this temperature stress if first exposed to 45°C for 0.5 to 4.5 h. Fat bodies from adult females produce a set of at least six specific polypeptides with molecular weights of 81, 73, 68, 42, 28, and 24×10³ in reponse to heat shock (39–47°C for 1.5 h). These molecular weights closely match those of the heat shock proteins (hsps) observed inDrosophila, with the possible exception of the 42 kd protein of locusts. The optimal temperature for induction of hsps in locusts is 45°C, which is one of the highest heat shock temperatures reported in metazoans. The correspondence between the optimal temperature for hsp induction and the temperature at which enhanced heat tolerance is acquired (both 45 °C) suggests that the hsps may be associated with thermal protection in these insects.There appears to be no substantial translational control in the locust heat shock response, since other proteins are produced, albeit with some reduction, under heat shock conditions. Vitellogenin synthesis in fat bodies at 45°C is 55% of that observed at 30°C. The high optimal heat shock induction temperature and the continued synthesis of non-heat shock proteins may be adaptive to the locust's natural environment.     

Temperature ranges over which rainbow trout fibroblasts survive and synthesize heat-shock proteins

Cultures of the rainbow trout fibroblast cell line RTG-2 withstood temperatures from 0 degrees C to 28 degrees C. At 0 degrees C and 28 degrees C, no proliferation occurred, but cells persisted for at least 7 days. If the cultures were placed back at 22 degrees C, proliferation returned to normal in those that had been kept at 0 degrees C but was reduced in cultures that had been kept at 28 degrees C. Above 28 degrees C, cultures survived for only short periods. If RTG-2 cells that were grown routinely at 22 degrees C were shifted to 26, 28, and 30 degrees C, heat shock proteins (hsps) of 100, 87, 70, 68, 60, 39, 27, and 19 kilodaltons were synthesized. Synthesis was most pronounced at 28 degrees C, and at this temperature hsp synthesis was maximal by 2 hr and had returned to control levels by 36 hr. Individual hsps were synthesized maximally at slightly different times and temperatures, but under all conditions hsps 87 and 70 were most abundant. If cultures were shifted to 24 degrees C or 32 degrees C, hsp synthesis was not observed. Neither the placement of cultures at 5 degrees C nor the shift of cultures that had been maintained at 0 degrees C or 5 degrees C back to 22 degrees C induced the synthesis of hsps. However, cultures incubated at 5 degrees C for 24 hr did synthesize hsps at 26 degrees C, 28 degrees C, and 30 degrees C.     

The effect of dormancy on the heat shock response in gladiolus cormels

Cormels of Gladiolus X gandavensis Van Houtte respond to heat shock by an induced synthesis of heat shock proteins. Synthesis of some of the non-heat shock proteins is concomitantly reduced. The ability of dormant cormels to synthesize heat shock proteins (hsps) and to repress the synthesis of non-hsps is greater than that of nondormant ones. A hsp of apparent molecular weight 68 kilodaltons is synthesized only in dormant cormels or in cormels that lost their dormancy after long storage at 25°C. The synthesis of hsps at 40°C, but not at 25°C, is promoted by abscisic acid in nondormant cormels. Methionine incorporation into hsps declines after a 4-hour incubation period at 40°C. Induction of hsps is stronger if exposure to extreme temperature is done gradually.