QUANTITATIVE AND QUALITATIVE ANALYSES OF PROTEIN SYNTHESIS DURING HEAT SHOCK IN THE MARINE DIATOM NITZSCHIA ALBA (BACILLARIOPHYCEAE)1

Protein synthesis in the diatom Nitzschia alba Lewin and Lewin was drastically altered when the cells were incubated at a supraoptimal temperaeture. Quantitatively, the overall protein synthesis was greatly suppressed as indicated by teh rate of [³⁵S] methionine incorporation. The extent of suppression of protein synthesis was proportional to the severity of the heat-shock treatment which was a combination of elevated temperature and treatment duration. The in viro synthesized proteins were also qualitativelty anlayzed by two-dimensional gel electrophoresis. Dependeing on the treatment condition, a set of heat-shock proteins (HSPs) were induced. They were best detected when the cells were subjected to shocks of 35°C for 60 min or 40°C for 10 min followed by a 60 min labelling at 30°C. The results revealed 16 HSps which had moluecular weights ranging from 24–94 kD and isoelectric points ranging from 5.50–7.10. Some of the HSps were identical in molelcular weights but with differeentr isoelectric points. The induction and accumulation of HSPs in Nitzschia alba were transitory. Prologned heat-shock treatments resulted in a complete cessation of protein syntehsis and no further induction of HSPs. In all aspects, the heat shock response of diatoms was similar to that in higher plants such as soybean, maize and tobacco but differenet from most animal systems.     

Heat shock proteins and effects of heat shock in plants

Soybean seedlings when exposed to a heat shock respond in a manner very similar to that exhibited by cultured cells, and reported earlier [2]. Maximum synthesis of heat shock proteins (HSPs) occurs at 40C. The heat shock response is maintained for a relatively short time under continuous high temperature. After 2.5 hr at 40 C the synthesis of HSPs decreases reaching a very low level by 6 hr. The HSPs synthesized by cultured cells and seedlings are identical and there is a large degree of similarity in HSPs synthesized between the taxonomically widely separated species, soybean and corn. Storage protein synthesis in the developing soybean embryo is not inhibited but is actually stimulated during a heat shock, unlike most other non-HSPs, whose synthesis is greatly reduced. Seedlings respond differently to a gradual increase in temperature than they do a sudden heat shock. There is an upward shift of several degrees in the temperature at which maximum protein synthesis occurs and before it begins to be inhibited. In addition, there appears to be a protection of normal protein synthesis from heat shock inhibition when the temperature increase is gradual. An additional function of the heat shock phenomenon might be the protection of seedlings from death caused by extreme heat stress. The heat shock response appears to have relevance to plants in the field.     

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.     

The heat-shock response in xenopus oocytes is controlled at the translational level

Xenopus laevis oocytes respond to high temperature (>31°C) by the synthesis of one major (70 kilodalton) protein and by a gradual reduction in the rate of normal protein synthesis. In contrast with most other cells, the heat-shock response of Xenopus oocytes is controlled exclusively at the translational level. Enucleated or α-amanitin-injected oocytes synthesize normal levels of heat-shock protein. Thus high temperature induces the translation of preformed heat-shock mRNA. This continues for more than a day after a shift back to a normal temperature, but ceases within 2 days. Heat-shock protein synthesis can be sequentially induced and inactivated in the same oocyte over several days. We conclude that an oocyte contains 10–100 pg of heat-shock mRNA, which is synthesized during oogenesis at the normal temperature, and which is stored in an inactive state by a “masking” mechanism.     

Heat shock response of Dictyostelium

In response to a shift from 22 to 30°C the relative rate of synthesis of a small number of proteins is dramatically increased in Dictyostelium discoideum. The cells neither grow nor develop at this temperature but die slowly with a half-life of 18 hr. The major protein synthesized in response to a heat shock to 30°C in either growing cells or developing cells has an apparent molecular weight of 70,000 (70K). An increase in the relative rate of synthesis of 70K can be seen as early as 20 min following heat shock. Synthesis of 70K remains high for 4 hr at 30°C and then decreases. Similar kinetics of 70K synthesis occur during recovery at 22°C following a 1-hr heat shock. RNA synthesis during the first half-hour of heat shock is essential for the high rate of 70K measured 2 hr later. By isoelectric focusing the 70K protein can be separated into two spots, one of which overlaps one of the major heat shock proteins of Drosophila melanogaster. The relative rate of synthesis of several other proteins (82K, 60K, 43K) increases less dramatically in Dictyostelium during heat shock at 30°C. A heat shock to 34°C results in rapid synthesis of these proteins but not of 70K. The relative rates of synthesis of most other proteins made at 22°C decreases, most notably that of actin. Synthesis of heat shock proteins at 30°C does not significantly affect viability at 30°C but dramatically prolongs the period of time the cells can survive at 34°C. Thus, 30°C appears to be a stasis condition for Dictyostelium which elicits a response essential for protection from lethal temperatures. The similarity of the heat shock response in Dictyostelium to that in Drosophila and vertebrate cells suggests that certain aspects of the response may be universal in eukaryotes.     

Differential responses of bovine oocytes and preimplantation embryos to heat shock

The authors sought to determine whether developmental differences in the magnitude of embryonic mortality caused by heat stress in vivo are caused by changes in resistance of embryos to elevated temperature. In this regard, responses of oocytes, two-cell embryos, four- to eight-cell embryos, and compacted morulae to heat shock were compared. An additional goal was to define further the role of cumulus cells and glutathione in thermoprotection of oocytes. In experiment 1, heat shock (41°C for 12 hr) decreased the number of embryos developing to the blastocyst stage for two-cell (26% vs. 0%) and four- to eight-cell (25% vs. 10%) embryos but did not affect morulae (37% vs. 42%). In experiment 2, exposure of two-cell embryos to 41°C for 12 hr reduced the number of four- to eight-cell embryos present 24 hr after the end of heat shock (88% vs. 62%). In experiment 3, heat shock reduced the number of two-cell embryos developing to blastocyst (49% vs. 8%) but did not affect subsequent development of oocytes when heat shock occurred during the first 12 hr of maturation (46% vs. 41% development to blastocyst); membrane integrity was not altered. In experiment 4, oocytes were cultured with an inhibitor of glutathione synthesis, _DL-buthionine-[S,R]-sulfoximine (BSO), for 24 hr and exposed to 41°C for the first 12 hr of maturation. Percentages of blastocysts were 35% (39°C), 18% (41°C), 17% (39°C+BSO), and 11% (41°C+BSO). For experiment 5, oocytes were either denuded or left with cumulus intact and were then radiolabeled with [³⁵S]methionine and [³⁵S]cysteine at 39°C or 41°C for 12 hr. Exposure of oocytes to 41°C for 12 hr reduced overall synthesis of ³⁵S-labeled TCA-precipitable intracellular proteins (18,160 vs. 14,594 dpm/oocyte), whereas presence of cumulus increased synthesis (9,509 vs. 23,246). Analysis by two-dimensional SDS PAGE and fluorography revealed that heat shock protein 68 (HSP68) and two other putative heat shock proteins, P71 and P70, were synthesized by all oocytes regardless of treatment. Heat shock did not alter the synthesis of HSP68 or P71 but decreased amounts of newly synthesized P70. Cumulus cells increased synthesis of P71 and P70. Results indicate there is a biphasic change in resistance to elevations in temperature as oocytes mature, become fertilized, and develop. Resistance declines from the oocyte to the two-cell stage and then increases. Evidence suggests a role for cumulus cells in increasing HSP70 molecules and protein synthesis. Data also indicate a role for glutathione in oocyte function. Mol Reprod Dev 46:138–145, 1997. © 1997 Wiley-Liss, Inc.     

Effect of cycloheximide or puromycin on induction of thermotolerance by sodium arsenite in Chinese hamster ovary cells: involvement of heat shock proteins

After sodium arsenite (100 microM) treatment, the synthesis of three major heat shock protein families (HSPs; Mr = 110,000, 87,000, and 70,000), as studied with one-dimensional gels, was enhanced twofold relative to that of unheated cells. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed as a 100,000-fold increase in survival from 10(-6) to 10(-1) after 4 hr at 43 degrees C, and as a thermotolerance ratio (TTR) of 2-3 at 10(-3) isosurvival for heating at 45.5 degrees C. Cycloheximide (CHM: 10 micrograms/ml) or puromycin (PUR: 100 micrograms/ml), which inhibited total protein synthesis and HSP synthesis by 95%, completely suppressed the development of thermotolerance when either drug was added after sodium arsenite treatment and removed prior to the subsequent heat treatment. Therefore, thermotolerance induced by arsenite treatment correlated with an increase in newly synthesized HSPs. However, with or without arsenite treatment, CHM or PUR added 2-6 hr before heating and left on during heating caused a 10,000-100,000-fold enhancement of survival when cells were heated at 43 degrees C for 4 hr, even though very little synthesis of heat shock proteins occurred. Moreover, these cells manifesting resistance to heating at 43 degrees C after CHM treatment were much different than those manifesting resistance to 43 degrees C after arsenite treatment. Arsenite-treated cells showed a great deal of thermotolerance (TTR of about 10) when they were heated at 45 degrees C after 5 hr of heating at 43 degrees C, compared with less thermotolerance (TTR of about 2) for the CHM-treated cells heated at 45 degrees C after 5 hr of heating at 43 degrees C. Therefore, there are two different phenomena. The first is thermotolerance after arsenite treatment (observed at 43 degrees C or 45.5 degrees C) that apparently requires synthesis of HSPs. The second is resistance to heat after CHM or PUR treatment before and during heating (observed at 43 degrees C with little resistance at 45.5 degrees C) that apparently does not require synthesis of HSPs. This phenomenon not requiring the synthesis of HSPs also was observed by the large increase in thermotolerance to 45 degrees C caused by heating at 43 degrees C, with or without CHM, after cells were incubated for 6 hr following arsenite pretreatment. For both phenomena, a model based on synthesis and redistribution of HSPs is presented.     

Analysis of the temperature-dependent temporal pattern of heat-shock-protein synthesis in fish cells

Continuous exposure of Chinook salmon embryo cells to an elevated incubation temperature of 24°C induces the transient expression of a set of heat-shock or stress proteins whereas maintenance of the cells at a higher incubation temperature of 28°C produces a continuous synthesis of these stress proteins. In vitro translation studies suggest that the temperature-dependent temporal pattern of stress-protein synthesis is correlated with the levels of stress-protein mRNA. This was verified using a recombinant-DNA probe complementary to the 70K heat-shock-protein mRNA. A transient increase in the level of the fish heat-shock 70K mRNA was observed in RNA samples isolated from cells continuously exposed at 24°C However, a constant increase in the level of this specific mRNA was found in RNA preparations obtained from cells maintained at 28°C Therefore, the temperature-dependent pattern of fish heat-shockprotein synthesis appears to be directly related to the level of heat-shock-protein mRNA.     

Heat shock modulates phosphorylation status and activity of nucleoside diphosphate kinase in cultured sugarcane cells

Nucleoside diphosphate kinase (NDPK) is involved in the regeneration of nucleoside triphosphates (NTPs) through its phosphotransferase activity via an autophosphorylating histidine residue. Additionally, autophosphorylation of serine and/or threonine residues is documented for NDPKs from various organisms. However, the metabolic significance of serine/threonine phosphorylation has not been well characterized. In this study we report the cloning and characterization of NDPKI from cultured sugarcane (Saccharum officinarum L. line H50-7209) cells, and modulation of serine autophosphorylation of NDPK1 in response to heat-shock (HS). Heat-shock treatment at 40°C for 2 h resulted in a 40% reduction in labeled phosphoserine in NDPK1. This dephosphorylation was accompanied by an increase in NDPK enzyme activity. In contrast, NDPK1 in cultured tobacco (cv. W-38) cells did not show changes in autophosphorylation or increased enzyme activity in response to HS. The mRNA or protein level of NDPK1 did not increase in response to HS. Sugarcane cells sustain the constitutive protein synthesis in addition to heat-shock protein synthesis during HS, while constitutive protein synthesis is significantly reduced in tobacco cells during HS. Thus, HS modulation of NDPK1 activity and serine dephosphorylation in sugarcane cells may represent an important physiological role in maintaining cellular metabolic functions during heat stress.     

Ethanol treatment triggers a heat shock-like response but no thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings

Non‐lethal heat‐shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat‐shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS‐like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non‐lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non‐lethal ethanol treatment. Pre‐treatment with a non‐lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non‐lethal HS pre‐treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance.     

Induction of Heat Shock Proteins and Acquisition of Thermotolerance in Germinating Pigeonpea Seeds

Heat shock proteins (HSPs) ranging in molecular masses from 14 to 110 kDa were induced in embryonic axes of germinating Cajanus cajan (L.) Millspaugh seeds after exposure to 40 °C for 1 or 2 h. At 45 °C, there was a marked decline in synthesis of HSPs. A close relationship was observed between HSPs induced and the growth of the germinating seeds. Pretreatment of germinating seeds at 40 °C for 1 h or 45 °C for 10 min followed by incubation at 28 °C for 3 h led to considerable thermotolerance (45 °C, 2 h) and the recovery of protein synthesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of a new high-temperature-induced 66-kDa heat-shock protein,antigenically related to heat-shock protein 72

M-14 human melanoma cells, following severe hyperthermic exposures, synthesized a heat-shock protein of 66 kDa (hsp 66), in addition to the major “classic” heat-shock proteins. This hsp 66 was not expressed following mild hyperthermic exposures sufficient to trigger the synthesis of the other heat-shock proteins. The induction of hsp 66 was observed also in Li human glioma cells treated at 45°C for 20 min. By contrast, hsp 66 was not induced in seven other human cell lines (both melanoma and nonmelanoma) when they were subjected to the same hyperthermic treatment. Immunological recognition experiments showed that hsp 66 cross-reacted with the inducible hsp 72, but not with the constitutive hsp 73. The possibility that hsp 66 is a breakdown product of hsp 72 was ruled out by the fact that Poly(A)⁺ RNA extracted from cells treated at 45°C for 20 min was able to direct the synthesis of hsp 66 (together with hsp 72) in a message-dependent rabbit reticulocyte lysate, as well as in microinjected Xenopus oocytes. By contrast, only the hsp 72 was expressed using Poly(A)⁺ RNA extracted from cells heated at 42°C for 1 h. Affinity chromatography experiments on ATP-agarose showed that hsp 66 did not bind ATP in vitro, hsp 66 was localized both in the cytoplasm (cytosol, mitochondria, and microsome fraction) and in the nuclei of cells recovered from a severe heat shock: this intracellular distribution closely corresponded to that of hsp 72. The nuclear-associated hsp 66 was found to be tightly bound to nuclear structures and could not be extracted by incubation in ATP-containing buffer. © 1996 Wiley-Liss, Inc.     

Lamin B is a prompt heat shock protein

The cellular response to hyperthermia involves the increased synthesis of heat shock proteins (HSPs) within several hours after treatment. In addition, a subset of proteins has been shown to be increased immediately after heating. These “prompt” HSPs are predominantly found in the nuclear matrix–intermediate filament fraction and are not present or detectable in unheated cells. Since the nuclear matrix has been suggested to be a target for heat-induced cell killing, prompt HSPs may play a prominent role in the heat shock response. Using Western blotting and flow cytometry, we found that an increase in the synthesis of lamin B, one of the major proteins of the nuclear lamina, is induced during heating at 45.5°C but not during heating at 42°C. Since it is an abundant protein which is constitutively expressed in mammalian cells, lamin B appears to be a unique member of the prompt HSP family. The kinetics of induction of lamin B during 45.5°C heating did not correlate with the dose-dependent reduction in cell survival. While increased levels of lamin B during 45.5°C heating do not appear to confer a survival advantage directly, a possible role for lamin B in cellular recovery after heat shock cannot be discounted. J Cell Physiol 178:28–34, 1999. © 1999 Wiley-Liss, Inc.     

Heat-Resistance and Heat-Shock Response in the Nosocomial Pathogen <Emphasis Type="Italic">Enterococcus faecium</Emphasis>

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°C, and no growth at 52°C or 55°C. In agreement, a marked decrease of general protein synthesis was observed at 52°C, and very light synthesis was detected at 55°C. The heat resistance of E. faecium cells was analyzed by measuring the survival at temperatures higher than 52°C and, after 2 h of incubation, viable cells were still observed at 70°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. Received: 29 March 2002 / Accepted: 5 July 2002     

Production of medium-chain-length hydroxyalkanoic acids from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> in pH stat

The production of β-glucuronidase (GUS) driven by the Arabidopsis small heat shock protein 18.2 promoter in liquid cultures of transgenic tobacco (Nicotiana tabacum) hairy roots is reported. Clone GD-3, showing high GUS heat induction and a moderate growth rate, was selected from 436 clones for study. Treatment of GD-3 with heat shock at 36–42°C for 2 h then recovery at 27°C resulted in an increase in GUS specific activity, while higher heat-shock temperatures led to a decline. These results were in accordance with the change in esterase activity, a measure of tissue viability. Using 2 h of 42°C heat shock and a recovery phase at 27°C, GUS specific activity increased rapidly and reached a maximum of 267.6 nmol 4-methylumbelliferyl β-D-glucuronic acid (MU) min⁻¹ mg⁻¹ protein at 24 h of recovery. When tissues were continuously heated at 42°C and tested without a recovery period, GUS mRNA was detectable at 2 h and peaked at 5 h, but GUS activity was not seen until 10 h and did not peak until 28 h; in addition, the maximum activity was lower than that seen after heat shock for only 30 min or 2 h, followed by recovery. This shows that recovery at normal temperature is crucial for the heat-inducible heterogeneous expression system of transgenic hairy roots. Multiple heat-shock treatments showed that this system was heat reinducible, although a gradual decline in GUS specific activity was seen in the second and third cycles.     

Recurrent heat shock impairs the proliferation and differentiation of C2C12 myoblasts

Heat-related illness and injury are becoming a growing safety concern for the farmers, construction workers, miners, firefighters, manufacturing workers, and other outdoor workforces who are exposed to heat stress in their routine lives. A primary response by a cell to an acute heat shock (HS) exposure is the induction of heat-shock proteins (HSPs), which chaperone and facilitate cellular protein folding and remodeling processes. While acute HS is well studied, the effect of repeated bouts of hyperthermia and the sustained production of HSPs in the myoblast-myotube model system of C2C12 cells are poorly characterized. In C2C12 myoblasts, we found that robust HS (43 °C, dose/time) significantly decreased the proliferation by 50% as early as on day 1 and maintained at the same level on days 2 and 3 of HS. This was accompanied by an accumulation of cells at G2 phase with reduced cell number in G1 phase indicating cell cycle arrest. FACS analysis indicates that there was no apparent change in apoptosis (markers) and cell death upon repeated HS. Immunoblot analysis and qPCR demonstrated a significant increase in the baseline expression of HSP25, 70, and 90 (among others) in cells after a single HS (43 °C) for 60 min as a typical HS response. Importantly, the repeated HS for 60 min each on days 2 and 3 maintained the elevated levels of HSPs compared to the control cells. Further, the continuous HS exposure resulted in significant inhibition of the differentiation of C2C12 myocytes to myotubes and only 1/10th of the cells underwent differentiation in HS relative to control. This was associated with significantly higher levels of HSPs and reduced expression of myogenin and Myh2 (P < 0.05), the genes involved in the differentiation process. Finally, the cell migration (scratch) assay indicated that the wound closure was significantly delayed in HS cells relative to the control cells. Overall, these results suggest that a repeated HS may perturb the active process of proliferation, motility, and differentiation processes in an in vitro murine myoblast-myotube model.     

Cell-free system derived from heat-shocked Escherichia coli: Synthesis of enzyme protein possessing higher specific activity

heat-shocked S30 extract (HS-S30 extract) was prepared from cells of Escherichia coli strain Q13 exposed to elevated temperatures (from 37°C to 42°C) for 30 min. In a cell-free system with HS-S30 extract, the synthesized CAT protein had higher specific activity than that synthesized by a cell-free system with S30 extract prepared from Q13 cells incubated at 37°C. SDS-PAGE analysis showed that the heat-shock proteins, GroEL and DnaK, which are known to be molecular chaperones, were significantly increased in the HS-S30 extract. The addition of GroEL or DnaK to the S30 extract system increased the specific activity of the synthesized CAT protein. Heat-shock induction thus offers an effective method of modifying E. coli cell extracts.     

Complexity and Genetic Variability of Heat-Shock Protein Expression in Isolated Maize Microspores

The expression of heat-shock proteins (HSPs) in isolated maize (Zea mays L.) microspores has been investigated using high-resolution two-dimensional electrophoresis coupled to immunodetection and fluorography of in vivo synthesized proteins. To this end, homogeneous and viable populations of microspores have been purified in sufficient amounts for molecular analysis from plants grown in controlled conditions. Appropriate conditions for thermal stress application have been defined. The analysis revealed that isolated microspores from maize display a classical heat-shock response characterized by the repression of the normal protein synthesis and the expression of a set of HSPs. A high complexity of the response was demonstrated, with numerous different HSPs being resolved in each known major HSP molecular weight class. However, the extent of this heat-shock response is limited in that some of these HSPs do not accumulate at high levels following temperature elevation. Comparative analysis of the heat-shock responses of microspores isolated from five genotypes demonstrated high levels of genetic variability. Furthermore, many HSPs were detected in microspores at control temperature, indicating a possible involvement of these proteins in pollen development at stages close to first pollen mitosis.