The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish,Carassius auratus

Goldfish Carassius auratus were acclimated to either 10 or 30°C for a minimum of 5 weeks. A 65-kDa protein specific to warm-temperature-acclimated fish was extracted from the gel with 70% formic acid after two-dimensional electrophoresis of the muscle cytoplasmic protein fraction. The 65-kDa protein thus prepared to homogeneity was used to raise specific antibodies in rabbit by conventional methods. The antibody produced exhibited specific reaction with a protein having the same molecular weight from brain and liver tissue, suggesting that the 65-kDa protein is a ubiquitous cytosolic component in warm-acclimated goldfish. When water temperature was increased from 20 to 30°C over a 20-h period, a prominent amount of the 65-kDa protein was observed in muscle tissue extracts within 5 days of additional rearing; this was demonstrated by immunoblotting with the specific antibody. The N-terminal amino acid sequence of the 65-kDa protein was determined as Asp-Glu-Pro-Gln-Gly-His-Gln-His (or Asp)-Glu-Leu, differing from that of a family of known heat-shock proteins having about 70 kDa in molecular mass (hsp 70). No interaction between ATP and the 65-kDa protein revealed by ATP-agarose affinity chromatography further confirmed the different properties of the 65-kDa protein from those of hsp 70.Abbreviations ATP adenosine 5-triphosphate - hsp heat-shock protein(s) - IgG immunoglobulin G - mRNA messenger ribonucleic acid - PMSF phenylmethylsulphonyl fluoride - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulphate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Heat sensitivity and protein synthesis during heat-shock in the tobacco hornworm,Manduca sexta

Summary Fifth instar larvae of the tobacco hornworm,Manduca sexta, tolerate 1-h exposures to temperatures as high as 42°C. Above 42°C, survival declines rapidly to 18% at 44°C and 0% at 48°C. As in other insects, the heat-shock response ofManduca sexta involves the induction of synthesis of heat-shock proteins very similar in size to theDrosophila heat-shock proteins (84, 73, 71, 27, 25, 23, and 22 kd). In the epidermis, heat-shock protein synthesis peaks at 42°C, correlating with the heat sensitivity of both the tissue itself and the intact larva. Some heat-shock proteins have different isoelectric forms depending on tissue. Also, the heat-shock proteins are synthesized over a wider range of temperatures in the imaginal discs and the fat body as compared to the epidermis. In contrast to dipteran insects,Manduca sexta does not exhibit a strong repression of non-heat-shock protein synthesis under tolerable conditions.Abbreviations TCA trichloroacetic acid - PAGE polyacrylamide gel electrophoresis - AZT arbitrary Zeitgeber time - kd kilodaltons     

Relationship between heat-shock protein synthesis and thermotolerance in rainbow trout fibroblasts

Summary The role of heat-shock protein synthesis in the development of thermotolerance by rainbow trout fibroblasts was examined. During the first 6 h after being shifted from 22°C to 28°C, cells of the rainbow trout fibroblast line, RTG-2, rapidly synthesized the major heat-shock proteins (hsps), hsps 87, 70 and 27, and developed tolerance to 32°C. After 24 h at 28°C hsp synthesis was drastically reduced but thermotolerance was maintained. If these thermotolerant cells were shifted to 32°C, hsp synthesis continued at a very low level, but if they were subsequently returned to 22°C, synthesis of hsps 70 and 27 was induced again. The addition of actinomycin D during the first 6 h at 28°C prevented hsp synthesis and the development of thermotolerance. The presence of actinomycin D during the incubation of thermotolerant cultures at 32°C blocked the reinitiation of hsps synthesis at 22°C but had no effect on survival. Therefore, the hsps that accumulated at 28°C were sufficient to allow cells to survive a subsequent thermal stress at 32°C.     

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.     

Induction of thermotolerance in potato microplants by acetylsalicylic acid and H2O2

Potato microplants propagated as nodal explants were subjected to heat treatments in vitro similar to those employed in the thermotherapy step of virus eradication procedures. Low concentrations (10^-6-10^-5 M) of acetylsalicylic acid (ASA) in the culture medium improved (by 3.7-fold) tolerance of a 5-week high-temperature (35C) treatment. Furthermore, tissues subcultured on to ASA-free medium following several weeks of growth on ASA were more thermotolerant (by 3.8-fold) of a 7 week 35C treatment, and (by 38-fold) of a 15 h 42°C heat-shock. Stems of microplants grown on ASA contained significantly less catalase activity and higher levels of H2O2 than controls. Explanting and heat treatment, however, reduced catalase activity to similar levels in ASA-treated and control microplant tissues. To investigate whether H2O2 could be involved in signal transduction during the induction of thermotolerance, nodal explants were incubated for 1 h in H2O2 (0.1-50 mM), and then cultured under standard conditions. The microplants that grew from the H2O2-treated explants showed concentration-dependent decreases in stem height, but were significantly more thermotolerant than controls, more than 1 month after the H2O2 treatment. Thus, thermotolerance induced in these conditions was extremely stable. It is concluded that both salicylate and H2O2 treatments can induced thermotolerance in this system.Keywords: Acetylsalicylic acid, heat-shock, hydrogen peroxide, potato, microplant, thermotolerance.      

Thermotolerance and HSP70 expression in the Mediterranean fruit fly Ceratitis capitata

The relationship between Hsp70 expression and thermotolerance has been well documented in Drosophila melanogaster. However, there is limited information on this relationship in other insect species. In this report we describe the Hsp70-thermotolerance relationship in one of the major fruit fly pests, Ceratitis capitata (medfly). Hsp70 expression and thermotolerance were assayed at a range of temperatures in several stages of medfly development. The most thermotolerant stage was found to be the late larval stage (100% survival at 41 °C) followed by adult flies and late embryos (100% survival at 39 °C). These three stages showed a positive relationship between Hsp70 expression and thermotolerance. Mid-larval and mid-embryonic stages were found less thermotolerant and the Hsp70-thermotolerance relationship was not evident. Early embryos did not express Hsp70 at any temperature and exhibited the lowest thermotolerance. The relationship between Hsp70 and inducible thermotolerance was also studied in late larvae. A pretreatment at 37-39 °C increased thermotolerance at higher temperatures by approximately 1 °C. In parallel, the pretreatment increased Hsp70 expression suggesting a close link between Hsp70 expression and inducible thermotolerance. The increased Hsp70 levels after pretreatment were found to be due to the increased levels of the hsp70 RNA.     

Heat-shock response in germinating pine pollen

Summary The in vitro culture of pine pollen at various temperatures reveals only a moderate degree of thermotolerance, with considerably reduced levels of growth at and above 35° C. Unlike the pollen of many previously studied species, pine pollen shows some ability to recover from short periods of growth at temperatures as high as 40° C, especially when such exposures occur during the early stages of pollen germination. The pollen of Pinus taeda, unlike that of most other species, shows both quantitative and qualitative changes in the proteins synthesized during germination in vitro following a switch to elevated temperatures (37° C). This response, which can be elicited both during the very early stages of germination as well as during the later stages of pollen tube growth, is reversible following a shift back to the lower temperatures. As previously shown with vegetative tissue of other plant species, the heat-shock response not only involves the induction of high-molecular-weight proteins (most notably 82 kDa and 70 kDa proteins), but also a number of low-molecular-weight (10–20 kDa) species. Two-dimensional gel electrophoretic analysis reveals a small number of qualitative differences in the types of low-molecular-weight heat-shock proteins synthesized in pollen versus vegetative tissue.     

Effect of Combined Salt and Heat Treatments on Germination and Heat-Shock Protein Synthesis in Lentil Seeds

The germination of lentil seeds was gradually reduced when seeds were exposed to temperature of 30 or 40 °C, either alone or combined with 0.1, 0.2 or 0.3 M NaCl or 34.1 % (m/v) PEG 8000, during 6 –12 h imbibition. [³⁵S]-methionine incorporation in 12 h imbibed lentil axes also decreased with increasing NaCl concentration at 20 and 40 °C, whereas at 30 °C only 0.3 M NaCl treatment partially inhibited protein synthesis. An analysis of newly synthesized proteins by 1-D SDS PAGE, showed that the expression of most polypeptides decreased following increasing stress. Among these, low molecular mass heat-shock proteins declining, higher in 40 °C treated axes than those treated at 30 °C, supports the hypothesis that at this temperature maximal level of expression of these proteins was achieved.     

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.     

Effect of constant temperatures on germination, radial growth and virulence of Metarhizium anisopliae to three species of African tephritid fruit flies

The effect of temperatureon conidial germination, mycelial growth, andsusceptibility of adults of three tephritidfruit flies, Ceratitis capitata(Wiedemann), C. fasciventris (Bezzi) andC. cosyra (Walker) to six isolatesof Metarhizium anisopliae were studied inthe laboratory. There were significantdifferences among the isolates in the effect oftemperature on both germination and growth.Over 80% of conidia germinated at 20, 25 and30°C, while between 26 and 67% conidiagerminated at 35°C and less than 10% at15°C within 24 hours. Radial growth was slowat 15°C and 35°C with all of theisolates. The optimum temperature forgermination and mycelial growth was 25°C. Mortality caused by the six fungal isolatesagainst the three fruit fly species varied withtemperature, isolate, and fruit fly species.Fungal isolates were more effective at 25, 30and 35°C than at 20°C. The LT₉₀values decreased with increasing temperature upto the optimum temperature of 30°C. Therewere significant differences in susceptibilitybetween fly species to fungal infection at allthe temperatures tested.     

Heat shock protein response to thermal stress in the Asiatic clam,Corbicula fluminea

A recent approach to evaluate environmenta induced damages has been damages has been suggested, based on the stress response. The approach involves the detection of stress protein induction in organisms to infer about environmental conditions in their surroundings. However, to be an indicator of adverse biological effects in the environment, the elevation of stress proteins should be compared to a response pattern for the experimental species. JuvenileCorbicula fluminea, collection from a control site, were submited to heat-shock stress in the laboratory to obtain the stress response pattern under normal and extreme conditions. Acclimated to 26°C, the specimens were submited to 29, 32, 35 and 38°C, for 96 h. After 1, 2, 4, 8, 24, 48, 72 and 96 h of exposure, clams were removed from each vial and prepared for stress protein analysis. Animals from the control site were frozen in liquid nitrogen at the time of collection, and prepared for stress protein analysis. Hsp60 and 70 were detected by immunoreactivity after separation on 12.5% polyacrylamide gels and transference to nitrocellulose by western blotting, to determine the stress protein concentrations. The result showed that hsp70 increased at 4h from the beginning of the experiment and progressed over the 96 h experimental period in animals exposed to 35°C. However hsp70 levels decreased between 4 h and 24 h for the clams stressed at their lethal temperature of 38°C. Immunoblotting with hsp60 showed similar reactivity. At 38°C there was an increase in the amount of hsp60 at 4h, reaching a maximum eight-fold level at 8h. By 96h, the amount decreased to levels lower than those observed at 4h. At 38°C the level of hsp60 began to decrease at 8 h and continue to decline to 24 h when the clams died. The data support the hypothesis of increasing concentrations of stress protein until the heat shock approaches the thermal limits for the species. The results of this research suggest the usefulness of using the stress response as a diagnostic in environmental toxicology. They confirm that the sps response may serve as a valid biomonitoring tool under chronic, sublethal exposures when it is still possible to prevent effects at organismal or higher organizational levels.     

Spinach leaf 70-kilodalton heat-shock cognate stabilizes bovine adrenal glucose-6-phosphate dehydrogenase in vitro without apparent stable binding

Spinach (Spinacia oleracea L.) leaf tissue 70-kilodalton heat-shock cognate was purified by ATP-agarose affinity and gel filtration. Gel filtration of the affinity-purified protein resolved it into three forms: monomer, dimer, and oligomer. In the absence of ATP, the majority of the heat-shock cognate existed as a monomeric form with lesser amounts of dimer and oligomer. Addition of 3 mM ATP to the purified protein, containing all three forms, converted the dimeric and monomeric forms to a high-molecular-weight complex. Removal of ATP from the complex by dialysis resulted in the reappearance of the dimeric and monomeric forms. Addition of ATP to the highly purified monomer had no effect on its gel-filtration migration. Neither purified monomeric or dimeric forms showed stable binding to denatured proteins; however, both forms of the purified heat-shock cognate were able to stabilize the enzymatic activity of bovine adrenal glucose-6-phosphate dehydrogenase over a 48-h period at 25° C. In addition, the activity of glucose-6-phosphate dehydrogenase in the presence of purified heat-shock cognate dimer or monomer could be rapidly decreased in an ATP-dependent fashion depending on the order of the substrate addition to the reaction mixture. Circular-dichroism studies indicated that addition of ATP to the spinach 70-kDa heat-shock cognate caused a conformation change from -helical to a greater -sheet content. How conformational character may influence the stabilizing activity of the heat-shock cognate in a mechanism which does not require stable peptide binding is discussed.Abbreviations BiP immunoglobulin binding protein - BSA bovine serum albumin - CA carbonic anhydrase - CD circular dichroism - G6PDH glucose-6-phosphate dehydrogenase - HSP heat shock protein - HSP70 70-kilodalton heat shock protein - HSC70 70-kilodalton heat-shock cognate - MDH malate dehydrogenase - PVDF polyvinylidene difluoride The authors wish to thank C. Kaye and R. Henry for their critical review and discussion of this paper. The antibody for tobacco BiP was the generous gift of J. Denecke. Financial support for this work was provided by the National Science Foundation DCB 9017625. This is Florida Agricultural Experiment Station Journal Series No. R-04110.     

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.     

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.     

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.     

Targeted Disruption of sti35, a Stress-Responsive Gene in Phytopathogenic Fungus Fusarium oxysporum

sti35 is one of the heat-shock genes in Fusarium oxysporum, which is a fungal pathogen for wilt disease in plants. We have isolated a genomic clone of sti35 and used it to create disruption mutations. Disruption of the sti35 coding region resulted in the loss of a 32-kDa protein present in heat-shocked cells. The disruption had no detectable effect on growth and development at various temperatures, nor on the ability to acquire thermotolerance in nutrient medium. But the sti35 disruptants showed increased thermotolerance, relative to the wild-type strain, when incubated in minimal medium after heat treatment. Received: 21 March 2000 / Accepted: 23 May 2000     

Differences in thermotolerance induced by heat or sodium arsenite: correlation between redistribution of a 26-kDa protein and development of protein synthesis-independent thermotolerance in CHO cells

In previous studies, we have demonstrated the differences in thermotolerance induced by heat and sodium arsenite (Lee et al., Radiat. Res. 121, 295-303, 1990). In this study, we investigated whether a 26-kDa protein might play an important role in evincing these differences. Chinese hamster ovary (CHO) cells treated for either 1 h with 100 microM sodium arsenite (ARS) or 10 min at 45.5 degrees C became thermotolerant to a test heat treatment at 43 degrees C administered 6 or 12 h later, respectively. After the test heating at 43 degrees C for 1.5 h, the level of 26-kDa protein in the nucleus was decreased by 92% in nonthermotolerant cells, 78% in ARS-induced thermotolerant cells, and 3% in heat-induced thermotolerant cells. Inhibiting protein synthesis with cycloheximide (CHM, 10 micrograms/ml) after ARS treatment eliminated thermotolerance to 43 degrees C and delayed restoration of the 26-kDa protein in the nucleus. In contrast, CHM neither prevented the development of thermotolerance nor inhibited the restoration of the 26-kDa protein in heat-induced thermotolerant cells. However, when cells were exposed to cold (4 degrees C), immediately after initial heating, restoration of the 26-kDa protein and development of thermotolerance did not occur. These results demonstrate a good correlation between the restoration and/or the presence of this 26-kDa protein and the development of protein synthesis-independent thermotolerance.     

Isolation of Arabidopsis mutants lacking components of acquired thermotolerance

Acquired thermotolerance is a complex physiological phenomenon that enables plants to survive normally lethal temperatures. This study characterizes the temperature sensitivity of Arabidopsis using a chlorophyll accumulation bioassay, describes a procedure for selection of acquired thermotolerance mutants, and provides the physiological characterization of one mutant (AtTS02) isolated by this procedure. Exposure of etiolated Arabidopsis seedlings to 48 degrees C or 50 degrees C for 30 min blocks subsequent chlorophyll accumulation and is eventually lethal. Arabidopsis seedlings can be protected against the effects of a 50 degrees C, 30-min challenge by a 4-h pre-incubation at 38 degrees C. By the use of the milder challenge, 44 degrees C for 30 min, and protective pretreatment, mutants lacking components of the acquired thermotolerance system were isolated. Putative mutants isolated by this procedure exhibited chlorophyll accumulation levels (our measure of acquired thermotolerance) ranging from 10% to 98% of control seedling levels following pre-incubation at 38 degrees C and challenge at 50 degrees C. The induction temperatures for maximum acquired thermotolerance prior to a high temperature challenge were the same in AtTS02 and RLD seedlings, although the absolute level of chlorophyll accumulation was reduced in the mutant. Genetic analysis showed that the loss of acquired thermotolerance in AtTS02 was a recessive trait. The pattern of proteins synthesized at 25 degrees C and 38 degrees C in the RLD and AtTS02 revealed the reduction in the level of a 27-kD heat shock protein in AtTS02. Genetic analysis showed that the reduction of this protein level was correlated with the acquired thermotolerance phenotype.     

Response and tolerance of toxigenic Vibro cholerae O1 to cold temperatures

Survival and tolerance at cold temperatures, the differentially expressed cellular proteins, and cholera toxin (CTX) production were evaluated in Vibrio cholerae O1. Rapid loss of culturability and change to distinct coccoid morphology occurred when cultures of V. cholerae O1 were exposed to 5°C directly from 35°C. Also, cultures of V. cholerae first exposed to 15°C for 2 h and then maintained at 5°C failed to exhibit an adaptive response, instead a rapid loss of viable plate count was noticed. Results from Western blot experiments revealed the absence of a major cold shock protein, CS7.4. Also, a decreased level of CTX was noticed in V. cholerae O1 cultures exposed to 5 or 15°C after first being exposed to 15°C for 2 h, followed by transfer to 5°C. Reduced expression of CTX at cold temperatures, compared to the cultures maintained at 35°C, may be a result of decreased cellular metabolic activity. When V. cholerae O1 cultures were exposed to 15°C for 2 h, elevated expressions of 8, 26 and 194 kDa, and decreased expression of 28 and 183 kDa proteins occurred. It is suggested that these differentially expressed cold-responsive proteins are involved in regulating culturability and conversion to a coccoid cell morphology in V. cholerae O1.