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.     

Multiple responses to heat stress by the basidiomyceteSchizophyllum commune

Temperatures in excess of 45°C are required to stop the growth ofSchizophyllum commune colonies. Transfer of colonies from normal growth conditions (21°C) to 55°C, while halting mycelial expansion and increasing the production of aerial hyphae, was not lethal. Shortterm heat shock (3h) resulted in the appearance of nine proteins resolvable by SDS-PAGE that were newly synthesized or had their synthesis increased. The molecular weights of these proteins qualify two of them as being members of the hsp90 and hsp 70 families of heat shock proteins. Heat shock also affected proteolytic processes in the colonies. Changes in the pattern of ubiquitinated protein conjugates occurred; fewer high-molecular-weight conjugates were found in heat-shocked colonies, and the appearance of a ladder of lower-molecular-weight conjugates was noted. Protease enzymes detected by gelatin-gel PAGE showed a general decrease in activity. One of these proteases, which was up-regulated during nitrogen deprivation, showed an intermediate response during the combined stresses of heat shock and nitrogen starvation.     

Effect of EGF on the intracellular distribution of heat-shock proteins in A431 cells

The intracellular distribution of hsp70 and hdj1 was studied using immunofluorescent method. In nonstimulated cells hsp70 and hdj1 were observed in the cytoplasm of A431 cells. When 100 ng/ml EGF was added for 15 min, both hsp70 and hdj1 were accumulated in the nuclei. Later on (up to 1 h) hsp70 was exported from the nuclei to be observed mainly in the cytoplasm, whereas hdj1 remained in the nuclei. In cells exposed to tyrphostin AG1478, this inhibitor of tyrosine kinase activity of EGF receptor prevented EGF-dependent accumulation of hsp70 and hdj1 in the nuclei. U73122, an inhibitor of phospholipase C activity, induced tyrosine phosphorylation of EGF receptor without EGF stimulation. In cells treated with U73122, both hsp70 and hdj1 were detected in the nuclei of non-stimulated cells. It is concluded that the intracellular distribution of heat shock proteins in A431 cells depends on tyrosine kinase activity of EGF receptor. Here we report for the first time the influence of EGF on the intracellular redistribution of heat shock proteins.     

TMV protein synthesis is not translationally regulated by heat shock

Summary Tobacco mosaic virus (TMV) protein synthesis in tobacco leaf tissue was not translationally regulated under conditions of heat shock as were most of the other proteins that were produced at 25°C. Upon shift from 25°C to 37–40°C, most host protein synthesis was inhibited followed by initiation of synthesis of heat shock proteins. In contrast, TMV protein synthesis continued after the temperature shift. This phenomenon allowed the enhancement of detection of TMV protein synthesis in tobacco leaves. The most prominent proteins labeled were viral when tissue was labeled during the first hr following the shift to 40°C, a period after heat shock repression of host protein synthesis, but before the onset of most heat shock protein synthesis. Another method to predominately label viral proteins was to incubate infected leaves for periods at 35°C which induced repression of preexisting host protein synthesis without inducing synthesis of heat shock proteins.     

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.     

Differential regulation of small heat-shock genes in plants: analysis of a water-stress-inducible and developmentally activated sunflower promoter

We have isolated two sunflower genes, Ha hsp 18.6 G2 and Ha hsp 17.7 G4, that encode small heat shock proteins (sHSPs). RNAse A protection experiments, carried out with RNA probes transcribed from each gene and hybridized to sunflower total RNA, allowed us to distinguish their mRNA accumulation patterns. In sunflower, Ha hsp 17.7 G4 mRNAs accumulated during zygotic embryogenesis at 25°C. In vegetative tissues, these mRNAs accumulated in response to either heat shock (42°C), abscisic acid (ABA), or mild water stress treatments. In all cases, the mRNAs were transcribed from the same initiation site. In contrast, Ha hsp 18.6 G2 mRNAs accumulated only in response to heat-shock. This result demonstrates differential regulation of these two sHSP genes. The complex regulation depicted by the Ha hsp 17.7 G4 promoter has been further analyzed in transgenic tobacco, using G4::GUS translational fusions. Developmental induction of Ha hsp 17.7 G4 during zygotic embryogenesis was faithfully reproduced in the transgenic plants. 5-distal sequences (between -1132 and -395) were required to confer a preferential spatial expression of GUS activity in the cotyledons. More proximal sequences (from -83 to +163) conferred to the chimeric genes most of the developmental regulation, and the responses to ABA and heat shock characteristic of the Ha hsp 17.7 G4 promoter. The water stress response of this gene was not reproduced in transgenic tobacco and, thus, could be uncoupled from its regulation during embryogenesis.     

Heat and cold shock responses in different strains of Thiobacillus ferrooxidans

Different strains of Thiobacillus ferrooxidans were examined for their ability to produce a heat shock and a cold shock response. Strain A1, heat shocked from 20° to 35°C, acquired thermotolerance, as it showed a 1000-fold reduction in cell mortality when exposed to the supermaximum temperature of 42°C, as compared to a non-heat-shocked control. A heat shock from 25° to 35°C yielded similar results, although a higher degree of thermotolerance was achieved for the shorter exposure times. Cultures heat shocked for 5 h showed a five-log reduction in viable counts after 41 h at 42°C, whereas non-heat-shocked cultures showed a similar reduction in viability in 28 h. Conferred thermotolerance was immediate and sustained for the duration of the exposure to 42°C. Heat-shocked cultures were not significantly protected against loss of viability due to freezing (-15°C for 24 h). Strain S2, cold shocked from 25° to 10°C, and strain D6, cold shocked from 25° to 5°C, were not protected against freezing at-15°C. An analysis of proteins extracted from heat-shocked cells of strain A1 showed the presence of at least one newly induced protein and eight hyper-induced proteins. The molecular weights of the heat shock proteins were in the range of 15–80.3 kDa.     

Nucleosomal organization of a BPV minichromosome containing a human H4 histone gene

Summary When the body temperature of rats is elevated to 42°C, four heat shock proteins, with the molecular weights of 70000, 71000, 85000, and 100000 (hsp 70, hsp 71, hsp 85, and hsp 100, respectively), are induced in various tissues of rats (Fujio et al., J Biochem 101, 181–187, 1987). Heat shock proteins are induced by various stresses other than heat in varieties of cultured cells, so we studied whether heat shock proteins are induced in intact rats by different treatments. Analysis of the translation products of poly(A) + RNA isolated from the livers of rats recovering from ischemia of the liver showed that mRNAs for hsp 70, hsp 71, and hsp 85 were induced. These hsp-mRNAs were also induced in the livers of rats 6 h after a partial hepatectomy, and had returned to control levels 24 h after the surgery. These results suggested that heat shock proteins have not only the function of protection against various stresses but also physiological functions in the normal growth and development of animals.     

Effect of ATP on the Release of hsp 70 and hsp 40 from the Nucleus in Heat-Shocked HeLa Cells

We have recently found a novel 40-kDa heat-shock protein (hsp 40) in mammalian and avian cells and reported that the N-terminal amino acid sequence of mammalian hsp 40 has homology with the bacterial DnaJ heat-shock protein. Also, hsp 40 has been shown to be translocated from the cytoplasm into the nuclei/nucleoli by heat shock and colocalized with hsc 70 (p73) in the nucleoli of exactly the same cells. We here investigated the effect of ATP on the release of hsp 70 (both constitutive p73 and inducible p72) and hsp 40 from the nuclei/nucleoli of heat-shocked HeLa cells which were permeabilized with Nonidet-P40 using immunoflourescence and immunoblotting. Hsp 70 in the nucleoli was released by the addition of ATP but not by ADP, GTP, nonhydrolyzable ATP, nor high salt buffer. In contrast, hsp 40 was not released from the nucleoli with any of these treatments or any combination of these treatments. Thus, hsp 40 might dissociate spontaneously from the nucleoli after hsp 70 has been released in an ATP-dependent manner. Using cell fractionation methods, we showed that while the majority of hsp 40 is localized in the cytoplasm, a small portion of it is located in the microsome fraction in non-heat-shocked control cells and in cells which recovered from heat shock.     

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.     

Chromatin-associated heat shock proteins of Dictyostelium

A heat shock response has been observed in a wide variety of eukaryotic organisms and may be universal. In Drosophila four heat shock proteins (hsp 22, 23, 26, and 27) have been found in nuclei (A. Arrigo, S. Fakan, and A. Tissieres, 1980, Develop. Biol. 78, 86–103). Eight heat shock-induced proteins of Dictyostelium discoideum were found to be preferentially localized in nuclei. They ranged in size from 26,000 to 32,000 daltons and could be recognized among the chromatin-associated proteins. Partial degradation of the chromatin released the low-molecular-weight heat shock proteins to the same extent as the histones. The heat shock response has been shown to result in protection of cells to the lethal effects of high temperature in a variety of organisms including Dictyostelium. We found that this response is extremely rapid in Dictyostelium being maximal by 30 min. The low-molecular-weight heat shock proteins enter the nuclei rapidly and so could play a role there in thermal protection. A mutant strain was isolated which is impaired in the protection afforded by a heat shock. This strain synthesizes most proteins normally but specifically fails to synthesize the low-molecular-weight heat shock proteins under conditions which result in their induction in wild-type cells.     

Synthesis of heat shock proteins inThermoanaerobacterium thermosulfurigenes EM1 (Clostridium thermosulfurogenes EM1)

The response to heat stress was examined inThermoanaerobacterium thermosulfurigenes EM1. Upon a temperature shift-up from 50° to 62°C, four heat shock proteins (hsps) were synthesized at an elevated level. Two proteins were found to be immunologically related to theEscherichia coli GroEL protein and theMycobacterium tuberculosis hsp71 (DnaK similar protein), and the correspondinggroE anddnaK homologous sequences were detected in the chromosome ofT. thermosulfurigenes EM1. The heat shock response in this thermophile was transient, with a maximum synthesis of hsps between 10 and 15 min after the shock. The enhanced synthesis of DnaK and GroEL was consistent with increased mRNA levels of the genes, which reached a maximum 15 min after heat treatment.     

Preliminary study of heat shock proteins in nemerteans

Nemerteans experience varying environmental temperatures during low tide exposures. Inducible heat shock proteins (hsps) have been reported for most organisms following both artificial heat stress and natural environmental temperature variations. This preliminary study reports the presence of hsps in the phylum Nemertea. A lethal temperature of 36 °C was determined for Paranemertes peregrina Coe, 1901. The nemerteans were exposed to a temperature of 34 °C for 2 h. After a 2 h recovery time, the worms were then analyzed for hsps by SDS–PAGE and western immunoblot protocols. Control worms were allowed to acclimate to ambient temperatures (13–15 °C) before hsp analysis. Hsp70 and hsp90 were detected in both the control and heat-shocked worms in highly variable concentrations, and the latter group had significantly elevated hsp70 levels. In addition, the analysis detected different isoforms of hsp70. The detection of hsps indicates a possible role in nemertean physiology during response to thermal stress, and potentially to other environmental challenges.     

Polyunsaturated fatty acids enhance the heat induced stress response in rainbow trout (Oncorhynchus mykiss) leukocytes

The heat shock response has been studied extensively, yet the molecular signals that trigger the response remain elusive. The dogma of the heat shock response contends that denatured proteins initiate the response, but evidence is accumulating to point to a more complex system in which at least more than one signal is involved in this process. Thermal stress initiates changes in cellular phospholipid membrane physical state, which when acted upon by phospholipases may release lipid mediators that could serve as triggering signals during the heat shock response. We have examined the heat shock response in freshly isolated leukocytes from the pronephros of rainbow trout (Oncorhynchus mykiss). In this study, we show that leukocytes isolated from rainbow trout acclimated to 5 or 19°C express elevated levels of heat shock protein 70 (hsp70) mRNA when heat shocked at 5°C above their respective acclimation temperature and supplementation with exogenous docosahexaenoic acid or arachidonic acid followed by heat shock enhanced levels of hsp70 mRNA. The time course for docosahexaenoic acid induced enhancement of hsp70 mRNA was accelerated compared with heat shock alone, and staurosporine inhibited the docosahexaenoic acid induced increase of hsp70 mRNA. We also provide evidence that phospholipase A₂ is involved in the heat shock response.