Thermotolerance and the heat-shock response in Candida albicans

At elevated temperatures, yeast cells of Candida albicans synthesized nine heat-shock proteins (HSPs) with apparent molecular masses of 98, 85, 81, 76, 72, 54, 34, 26 and 18 kDa. The optimum temperature for the heat-shock response was 45 degrees C although HSPs were detected throughout the range 41-46 degrees C. Protein synthesis was not observed in cells kept at 48 degrees C. Yeast cells survived exposure to an otherwise lethal temperature of 55 degrees C when they had previously been exposed to 45 degrees C. The thermotolerance induced during incubation at 45 degrees C required protein synthesis, since protection was markedly reduced by trichodermin. Mercury ions induced a set of three stress proteins, one of which corresponded in size to an HSP, and cadmium ions evoked one stress protein seemingly unrelated to the HSPs observed after temperature shift.     

Synthesis, modification and structural binding of heat-shock proteins in tomato cell cultures

Synthesis of about 30 acidic and 18 basic heat-shock proteins (hsps) is induced in suspension cultures of tomato (Lycopersicon peruvianum) if subjected to supraoptimal temperature conditions (35-40 degrees C). A characteristic aspect of the plant heat-shock response is the formation of cytoplasmic granular aggregates, heat-shock granules, containing distinct heat-shock proteins as major structural components and, in addition, several hitherto undetected minor acidic and basic heat-shock proteins. Structural binding of heat-shock proteins, i.e. assembly of heat-shock granules, is dependent on the persistance of supraoptimal temperature conditions. Despite the ongoing synthesis also at 25 degrees C, e.g. in pulse heat-shocked cultures, these proteins are accumulated exclusively in soluble form. Individual heat-shock proteins are characterized by their kinetics of synthesis and are classified by their compartmentation behaviour into class A proteins (exclusively found in soluble form, e.g. hsps 95 and 80), class B proteins (5-10% bound to heat-shock granules, e.g. hsps 70, 68), class C proteins (30-80% bound to heat-shock granules, e.g. hsps 21, 17, 15) and class D proteins, which are minor heat-shock proteins only detected in structure-bound form. Major representatives are modified proteins, i.e. hsps 95, 80, 70 and 68 are phosphorylated and hsps 80, 74, 70 and 17 are methylated proteins (numbers 70, 80 etc. refer to 10(-3) Mr). Under heat-shock conditions synthesis of the proteins detected in control cells (25 degrees C proteins) exhibits two patterns. There are proteins with continued and proteins with discontinued synthesis. Synthesis of most of the latter proteins is resumed very rapidly after shift-down to 25 degrees C, even in the presence of actinomycin D. We conclude that reversible segregation of distinct mRNA species from the translation apparatus contributes to the heat-shock-specific pattern of protein synthesis in plants also.     

Heat-shock protein synthesis in Chlamydomonas reinhardi. Translational control at the level of initiation of a poly(A)-rich-RNA coded 22-KDa protein in a cell-free system

The effect of temperature on the in vitro translation of control and heat-shock poly(A)-rich RNA, obtained from Chlamydomonas reinhardi cells, incubated for 2 h at 25 degrees C respectively, was studied using the wheat-germ translation system. Incubation of the cells at 42 degrees C induces the synthesis of RNAs coding for several heat-shock proteins, including a 22-kDa major polypeptide as well as several proteins of 45-94 kDa, as demonstrated by run-off translation of polyribosomes isolated from intact cells. However, the high-molecular-mass heat-shock proteins are poorly translated in the wheat-germ system. The poly(A)-rich RNA coding for the 22-kDa heat-induced polypeptide has an apparent sedimentation coefficient higher than that expected from the molecular mass of its translation product, and was preferentially translated in vitro at temperatures above 31 degrees C as compared with pre-existing RNAs. Raising the temperature of translation, slightly inhibited (10%) the runoff translation of polyribosomes isolated from intact cells. However, when initiation was carried out in vitro for a short time at increasing temperatures and translation continued at 25 degrees C in the presence of aurintricarboxylic acid, the 22-kDa heat-shock polypeptides was preferentially translated. Aurintricarboxylic acid did not significantly inhibit incorporation of [35S]methionine when added to polyribosomes isolated from control or heat-shocked cells. From the above data we conclude that the translation of the 22-kDa heat-shock protein is controlled in vitro at the initiation level.     

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.     

Detection of mRNAs coding for translationally regulated heat-shock proteins in non-heat-shocked thymic lymphocytes

Heat shock induces 31 proteins in thymic lymphocytes in 1 h, 11 of which are not blocked by cordycepin, suggesting that their induction may be regulated at the level of translation (Maytin, E.V., Colbert, R.A., and Young, D.A. (1985) J. Biol. Chem. 260, 2384-2392). The possibility that mRNAs coding for these 11 cordycepin-insensitive heat-shock proteins would be found in non-heat-shocked thymus cells was investigated. Analysis of 1500 in vitro translation products separated by giant two-dimensional gel electrophoresis revealed that poly(A)+ RNA isolated from non-heat-shocked thymus cells coded for proteins corresponding to 10 of the 11 non-cordycepin-inhibitable heat-shock proteins. Comparison of the relative rates of synthesis of these 10 proteins in whole cells incubated at 37 and 42 degrees C, with their synthesis in vitro directed by poly(A)+ RNA isolated from cells incubated at 37 degrees C, suggests that mRNAs for 7 of them are present in sufficient amounts in non-heat-shocked cells to account for their increased synthesis during heat shock. These results indicate that part of the response of thymic lymphocytes to heat shock involves a rapid increase in the translation of a group of pre-existing mRNAs that are normally translated at very low rates or not at all.     

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.     

Defect in expression of heat-shock proteins at high temperature in xthA mutants.

Escherichia coli mutants lacking exonuclease III (xthA) are defective in the induction of heat-shock proteins upon severe heat-shock treatment (upshift from 30 to 50 degrees C) but not mild heat-shock treatment (upshift from 30 to 42 degrees C). We show that this defect is due to the xthA mutation by complementation. Furthermore, increasing the gene dosage of xthA+ prolongs the synthesis of heat shock proteins seen after a shift to 42 degrees C. Increasing the gene dosage of htpR+ partially suppresses the defect of xthA mutants in the synthesis of heat-shock proteins at 50 degrees C. When an xthA strain was incubated at 42 degrees C before a shift to 50 degrees C, it was then able to carry out the synthesis of heat-shock proteins at 50 degrees C.     

Changes in intracellular levels of Ap3A and Ap4A in cysts and larvae of Artemia do not correlate with changes in protein synthesis after heat-shock.

Artemia larvae respond to a brief heat-shock between 28 degrees and 40 degrees C with an increase in the synthesis of two groups of proteins of Mr 68,000 and 89,000. At 40 degrees C synthesis of all other proteins is strongly repressed. Cysts, which are naturally thermotolerant, synthesise both heat-shock proteins at temperatures up to 47 degrees C but maintain normal protein synthesis. During pre-emergence development, Ap3A is present in cysts at a concentration twice that of Ap4A. The maximum level of 7.6 pmol/10(6) cells is reached shortly before hatching of the larvae. After hatching, the levels of both nucleotides decline. A 40 degrees C heat-shock produces a 1.8-fold increase in both nucleotides within 20 min in cysts and larvae. A 2.8-fold increase results from a 47 degrees C heat-shock to cysts. The rates of increase parallel but do not precede the increases in the heat-shock proteins. Since non-heat-shocked cysts possess higher levels of Ap3A and Ap4A than do heat-shocked larvae, the observed heat-induced changes in gene expression cannot be explained simply in terms of the intracellular concentrations of these nucleotides.     

Heat-shock response in Xenopus oocytes during meiotic maturation and activation

After a 60 min heat-shock at 36 degrees C, Xenopus oocytes are still able to accomplish a complete meiotic maturation in response to a progesterone treatment. The 36 degrees C heat-shock applied to maturing oocytes strongly enhances the synthesis of a single heat-shock protein of approx. 70 000 molecular weight (hsp70); after activation with the Ca2+-ionophore A 23187, matured oocytes still display the ability to synthesize hsp70 and to survive a heat-shock. A cycloheximide treatment combined with a heat-shock induces, during the recovery period, the synthesis of two heat-shock proteins, of approx. 70 000 and 83 000 molecular weight.     

Adaptive cellular response to hyperthermia: 31P-NMR studies

Dynamic intracellular ATP and Pi levels were measured non-invasively for Chinese hamster V79 cells by 31P-NMR under conditions of thermotolerance and heat-shock protein induction. High densities of cells were embedded in agarose strands, placed within a standard NMR sample tube, and perfused with medium maintained either at 37 or 43 degrees C at pH 7.35. Cell survival and heat-shock protein synthesis were assessed either from parallel monolayer cultures or cells dislodged from the agarose strands post-treatment. Thermotolerance (heat resistance) and heat-shock protein synthesis was induced by a 1 h exposure to 43 degrees C followed by incubation for 5 h at 37 degrees C. After the 5 h incubation at 37 degrees C, marked thermal resistance was observed in regard to survival with concomitant synthesis of two major heat-shock proteins at 70 and 103 kDa. Studies were also conducted where tolerance and heat-shock protein synthesis were partially inhibited by depletion of cellular glutathione (GSH) prior to and during heat treatment. Dynamic measurement of intracellular ATP of cells heated with or without GSH depletion revealed no change in steady-state levels immediately after heating or during the 5 h post-heating incubation at 37 degrees C where thermotolerance and heat-shock proteins develop. These data are consistent with other reported data for mammalian cells and indicate that the steady-state ATP levels in mammalian cells remain unchanged during and after the acquisition of the thermotolerant state.     

Characterization of the thermotolerant cell. I. Effects on protein synthesis activity and the regulation of heat-shock protein 70 expression

Exposure of mammalian cells to a nonlethal heat-shock treatment, followed by a recovery period at 37 degrees C, results in increased cell survival after a subsequent and otherwise lethal heat-shock treatment. Here we characterize this phenomenon, termed acquired thermotolerance, at the level of translation. In a number of different mammalian cell lines given a severe 45 degrees C/30-min shock and then returned to 37 degrees C, protein synthesis was completely inhibited for as long as 5 h. Upon resumption of translational activity, there was a marked induction of heat-shock (or stress) protein synthesis, which continued for several hours. In contrast, cells first made thermotolerant (by a pretreatment consisting of a 43 degrees C/1.5-h shock and further recovery at 37 degrees C) and then presented with the 45 degrees C/30-min shock exhibited considerably less translational inhibition and an overall reduction in the amount of subsequent stress protein synthesis. The acquisition and duration of such "translational tolerance" was correlated with the expression, accumulation, and relative half-lives of the major stress proteins of 72 and 73 kD. Other agents that induce the synthesis of the stress proteins, such as sodium arsenite, similarly resulted in the acquisition of translational tolerance. The probable role of the stress proteins in the acquisition of translational tolerance was further indicated by the inability of the amino acid analogue, L-azetidine 2-carboxylic acid, an inducer of nonfunctional stress proteins, to render cells translationally tolerant. If, however, analogue-treated cells were allowed to recover in normal medium, and hence produce functional stress proteins, full translational tolerance was observed. Finally, we present data indicating that the 72- and 73-kD stress proteins, in contrast to the other major stress proteins (of 110, 90, and 28 kD), are subject to strict regulation in the stressed cell. Quantitation of 72- and 73-kD synthesis after heat-shock treatment under a number of conditions revealed that "titration" of 72/73-kD synthesis in response to stress may represent a mechanism by which the cell monitors its local growth environment.     

Heat-resistance and heat-shock response in the nosocomial pathogen Enterococcus faecium

We have characterized the heat-shock response of the nosocomial pathogen Enterococcus faecium. The growth of E. faecium cells was analyzed at different temperatures; little growth was observed at 50 degrees C, and no growth at 52 degrees C or 55 degrees C. In agreement, a marked decrease of general protein synthesis was observed at 52 degrees C, and very light synthesis was detected at 55 degrees C. The heat resistance of E. faecium cells was analyzed by measuring the survival at temperatures higher than 52 degrees C and, after 2 h of incubation, viable cells were still observed at 70 degrees C. By Western blot analysis, two heat-induced proteins were identified as GroEL (65 kDa) and DnaK (75 kDa). Only one isoform for either GroEL or DnaK was found. The gene expression of these heat-shock proteins was also analyzed by pulsed-labeled experiments. The heat-induced proteins showed an increased rate of synthesis during the first 5 min, reaching the highest level of induction after 10 min and returning to the steady-state level after 20 min of heat treatment.     

Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.     

Studies of protoplast fusion in Streptomyces chrysomallus

Conditions for the regeneration of cells from protoplasts of Streptomyces chrysomallus, a producer of the peptide antibiotic actinomycin, are described. Regeneration of fusion products was most efficient at 27-30 degrees C on regeneration R2 medium (Okanishi et al., 1974) containing 0.25 M-sucrose. The addition of phosphate (150-300 mg 1(-1) to the medium and incubation at 23 degrees C proved to be optimal for the regeneration of individual strains. Highest recombination frequencies after protoplast fusion were obtained by fusing protoplasts in the presence of 45% (w/v) polyethylene glycol 6000. With strains that produce no, or little antibiotic, protoplasts must be present in excess in fusion mixtures in order to overcome inhibition of regeneration by the antibiotic-producing partner.     

The Anton blood group antigen is the erythrocyte receptor for Haemophilus influenzae

Abstract The protein synthesis pattern was investigated in Bacillus subtilis relA ⁺ and relA after heat shock using the highly sensitive 2-dimensional O'Farrell technique [1]. The synthesis of several proteins is markedly enhanced upon temperature shift-up in both strains. At 52°C the growth rate is drastically diminished because the synthesis of cellular proteins is inhibited. However, the production of heat-shock proteins is maintained. The synthesis of some of these presumptive heat-shock proteins is stimulated at 37°C in cells treated with H₂O₂ as well as with norvaline, which induces a guanosine tetraphosphate (ppGpp)-dependent stringent response.     

Transcription of the phosphoglycerate kinase gene of Saccharomyces cerevisiae increases when fermentative cultures are stressed by heat-shock

The single gene for phosphoglycerate kinase (PGK) in the haploid genome of Saccharomyces cerevisiae is expressed to a very high level in cultures fermenting glucose. Despite this it responds to heat-shock. When S. cerevisiae growing exponentially on glucose media was shifted from 25 degrees C to 38 degrees C transient increases of 6-7-fold in cellular PGK mRNA were observed. This elevation in PGK mRNA still occurred in the presence of the protein-synthesis inhibitor cycloheximide, but was not observed in cells bearing the rna1.1 mutation. From the kinetics of continuous labelling of PGK mRNA, relative to the labelling of other RNAs in the same cultures whose levels do not alter with heat-shock, it was shown that the elevation in PGK mRNA in response to temperature upshift reflects primarily an increased synthesis of this mRNA and not an alteration of its half-life. PGK mRNA synthesis is therefore one target of a response mechanism to thermal stress. Synthesis of PGK enzyme in glucose-grown cultures is efficient after mild (25 degrees C to 38 degrees C) or severe (25 degrees C to 42 degrees C) heat-shocks. Following the severe shock, the synthesis of most proteins is abruptly terminated, but synthesis of PGK and a few other glycolytic enzymes continues at levels comparable to the levels of synthesis of most of those proteins dramatically induced by heat (heat-shock proteins). Cells that overproduce PGK due to the presence of multiple copies of the PGK gene on a high-copy-number plasmid continue their overproduction of this enzyme during severe thermal stress. Therefore PGK mRNA is both elevated in level in response to heat-shock and translated efficiently at supra-optimal temperatures.     

A cya deletion mutant of Escherichia coli develops thermotolerance but does not exhibit a heat-shock response

An adenyl cyclase deletion mutant (cya) of E. coli failed to exhibit a heat-shock response even after 30 min at 42 degrees C. Under these conditions, heat-shock protein synthesis was induced by 10 min in the wild-type strain. These results suggest that synthesis of heat-shock proteins in E. coli requires the cya gene. This hypothesis is supported by the finding that a presumptive cyclic AMP receptor protein (CRP) binding site exists within the promoter region of the E. coli htpR gene. In spite of the absence of heat-shock protein synthesis, when treated at 50 degrees C, the cya mutant is relatively more heat resistant than wild type. Furthermore, when heat shocked at 42 degrees C prior to exposure at 50 degrees C, the cya mutant developed thermotolerance. These results suggest that heat-shock protein synthesis is not essential for development of thermotolerance in E. coli.     

Down-regulation of IL-1 beta biosynthesis by inducers of the heat-shock response

The effect of heat on IL-1 beta biosynthesis was investigated in both THP-1 cells, a myelomonocytic cell line which can be induced to make IL-1 alpha and beta, and human peripheral blood adherent monocytes (PBMC). Induction of THP-1 cells with LPS at 39 to 41 degrees C for 2 to 4 h resulted in the expected increased synthesis of the heat-shock proteins hsp 70 and hsp 90 but decreased synthesis of the IL-1 beta precursor protein, p35 (and its mRNA), compared with control cells at 37 degrees C. This appeared to be a direct effect on p35 synthesis rather than a block in LPS induction because heat also acted on preinduced cells. PBMC similarly incubated for 4 h with LPS required a temperature of 41 to 42 degrees C to induce hsp and show a decrease in p35 synthesis. Chemical inducers of the heat-shock response (heavy metals, sulphydryl reagents) were also effective inhibitors of IL-1 beta biosynthesis. A correlation was seen between the extent of IL-1 beta reduction and the level of hsp induction by chemical inducers in both THP-1 cells and PBMC which suggests that the two responses are linked. In addition, a gold salt currently used for therapy of chronic inflammation, auranofin, induced hsp and inhibited IL-1 beta biosynthesis, whereas a second salt, sodium aurothiomalate, did neither. These results support the hypothesis that elevated temperature is one of the physiologic signals for down-regulation of IL-1 beta biosynthesis through a mechanism related to the induction of hsp.