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Discordant expression of heat shock protein mRNAs in tissues of heat-stressed rats.

Although the induction of heat shock proteins (HSP) has been studied extensively in cultured cells, comparatively few studies have examined their expression in vivo. In this report, mRNA expression of two HSP families, HSP70 and HSP27, was investigated in brain, liver, lung, and skin of rats exposed to elevated ambient temperatures. The time course and relative magnitude of the heat-induced expression for these two HSP differed between tissues of the same animal. Even within the same tissue, HSP70 and HSP27 displayed differential kinetics of induction. In brain, lung, and skin, induction of HSP70 was dependent on the duration and temperature of the heat stress. This induction was transient with maximal HSP70 expression occurring at 1 h and returning to baseline 3 h after removal of the animals from heat stress. In liver, HSP70 expression did not show a direct relationship with temperature conditions and maximal induction did not occur until 6 h after heat stress. Heat-induced HSP27 expression was dependent on time and temperature of exposure in lung and skin but not in brain and liver. These findings demonstrate that the heat shock response in vivo lacks much of the coordinate control of expression characteristic of cultured cell populations and suggest that mechanisms controlling this cellular stress response are influenced by physiologic factors that cannot be studied in vitro.

Animals↗

Regulation of the synthesis of heat-shock proteins in heat-resistant variants of Chinese hamster fibroblasts.

The synthesis of the major heat-shock proteins (hsp) was compared in normal and heat-resistant Chinese hamster fibroblasts which express higher levels of the 70 kDa heat-shock protein (hsp70). Following exposure to a variety of experimental conditions that induce the elevated synthesis of the hsp, higher relative levels of hsp70 and lower relative levels of hsp89 and hsp110 were found in the heat-resistant variants. This effect was observed with all inducers tested. The relatively greater synthesis of hsp70 and relatively lower synthesis of hsp89 occurred at all temperatures tested and was found to be independent of cell culture conditions. The relatively greater increase in the levels of hsp70 in the heat-resistant variants after a mild heat shock was found to be a reflection of elevated levels of messenger RNA coding for this polypeptide. These results indicate that the heat-shock response in mammalian cells displays coordinate regulatory features and that the alteration of the expression of one of the hsp may affect the expression of the others.

Adaptation, Physiological↗

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.

Chaperonin 60↗

A heat-activated MAP kinase in tomato: a possible regulator of the heat stress response.

Adaptation to elevated temperatures is of major importance for the survival of plants. The role of kinases in heat stress response was studied in tomato by in gel and in solution kinase assays using myelin basic protein as substrate. The application of heat stress in a naturally occurring temperature range resulted in a fast and transient activation of a 50 kDa mitogen-activated protein (MAP) kinase both in a photoautotrophic cell suspension culture and in leaves of mature plants. The heat activation of the MAP kinase was shown to be calcium-dependent. The specific phosphorylation of tomato heat stress transcription factor HsfA3 by a partially purified preparation of the heat-activated MAP kinase supports a physiological role of the identified kinase activity in transducing the heat stress signal.

Calcium↗

Concomitant alterations in distribution of 70 kDa heat shock proteins, cytoskeleton and organelles in heat shocked 9L cells.

Maintenance of cell architecture and positioning of organelles are major functions of the cytoskeleton. On the other hand, induction of heat shock proteins (HSPs) and reorganization of the cytoskeleton are the most significant changes in heat-shocked mammalian cells. We examine the alterations in HSP70 and its constitutively expressed cognate, HSC70, as well as the cytoskeleton and organelles in 9L rat brain tumor cells upon heat shock. We employed fluorescence microscopy and scanning electron microscopy to follow these changes. Levels of HSP70s were quantified by Western blotting. Accumulation of HSC70 was more transient and the protein translocated to and subsequently exited from the nucleus more rapidly than HSP70. Changes in actin microfilaments include the nuclear localization of actin fraction and disappearance of cytoplasmic microfilament bundles, while the cortical actin microfilaments were almost unaffected. Furthermore, microtubules retracted slightly from the cell periphery but remained largely unchanged. In contrast, the intermediate filaments collapsed into the perinuclear region. The mitochondria converted from filamentous into granular forms and clustered in a region overlapping with the collapsed intermediate filaments. All of the above alterations are reversible and largely reverted after 8 h of recovery. The effect on Golgi organization was very transient and the apparatus assumed a normal appearance within 4 h after the heat treatment. The ER, on the other hand, was totally unaffected by the heat treatment. These observations help correlate the sequential events following a stress like heat shock and suggest possible physiological functions of these essential constituents of a cell under stress.

Animals↗

Stress-mediated signaling in PC12 cells - the role of the small heat shock protein, Hsp27, and Akt in protecting cells from heat stress and nerve growth factor withdrawal.

We have investigated the role of stress-activated signaling pathways and the small heat shock protein, Hsp27, in protecting PC12 cells from heat shock and nerve growth factor (NGF) withdrawal-induced apoptosis. PC12 cells and a stable cell line overexpressing Hsp27 (HSPC cells) were subjected to heat shock. This resulted in the rapid activation of Akt followed by p38 mitogen-activated protein kinase (MAPK) signaling, with phosphorylation and intracellular translocation of Hsp27 also detectable. Hsp27 was found to form an immunoprecipitable complex with Akt and p38 MAPK in both non-stimulated and heat shocked cells, although after heat shock there was a gradual dissociation of Akt and p38 from the Hsp27. Cells were differentiated with NGF and then subjected to NGF withdrawal, a treatment which results in substantial cell death over 24-72 h. Hsp27 was shown to be protective against this treatment, since HSPC cells which overexpress Hsp27 showed significantly less cell death than the parental PC12 cells. In addition, we observed that phosphorylation of Akt was maintained in HSPC cells subjected to heat shock and NGF withdrawal compared with the parental cells. Taken together, our results suggest that Hsp27 may protect Akt from dephosphorylation and may also act in stabilizing Akt.

Active Transport, Cell Nucleus↗

Autoregulation of the Escherichia coli heat shock response by the DnaK and DnaJ heat shock proteins.

All organisms respond to various forms of stress, including heat shock. The heat shock response has been universally conserved from bacteria to humans. In Escherichia coli the heat shock response is under the positive transcriptional control of the sigma 32 polypeptide and involves transient acceleration in the rate of synthesis of a few dozen genes. Three of the heat shock genes--dnaK, dnaJ, and grpE--are special because mutations in any one of these lead to constitutive levels of heat shock gene expression, implying that their products negatively autoregulate their own synthesis. The DnaK, DnaJ, and GrpE proteins have been known to function in various biological situations, including bacteriophage lambda replication. Here, we report the formation of an ATP hydrolysis-dependent complex of DnaJ, sigma 32, and DnaK proteins in vitro. This DnaJ-sigma 32-DnaK complex has been seen under different conditions, including glycerol gradient sedimentation and co-immunoprecipitation. The DnaK and DnaJ proteins in the presence of ATP can interfere with the efficient binding of sigma 32 to the RNA polymerase core, and are capable of disrupting a preexisting sigma 32-RNA polymerase complex. Our results suggest a possible mechanism for the autoregulation of the heat shock response.

Adenosine Triphosphate↗

Tissue-specific expression of zebrafish (Danio rerio) heat shock factor 1 mRNAs in response to heat stress.

All organisms respond to environmental, chemical and physiological stresses by enhanced synthesis of an evolutionarily conserved family of proteins known as heat shock proteins (HSPs) or stress proteins. Certain HSPs are also expressed constitutively during cell growth and development, and they function as molecular chaperones. The transcriptional regulation of hsp genes is mediated by the heat shock transcription factor (HSF). The stress response has been studied mostly in mammalian cell lines or organisms normally maintained under constant laboratory conditions. There is much less information on the regulation of the stress response of animals, such as fish, that have to tolerate large fluctuations in environmental and internal conditions. To characterize the regulation of the heat shock response in fish, we have cloned the first heat shock transcription factor from fish, zebrafish Danio rerio. Phylogenetic analysis confirms that the isolated zebrafish HSF belongs to the HSF1 family and is therefore designated zHSF1. Analysis by reverse transcriptase polymerase chain reaction (RT-PCR) shows the presence of two zHSF1 mRNA forms that are expressed in a tissue-specific fashion upon exposure to heat stress. Both forms are expressed in gonads under all conditions; in liver and to a lesser extent in the gills, the longer splice form of zHSF1 disappears upon heat shock. We present evidence for a unique tissue-specific regulation of HSF1 upon exposure to elevated temperature.

Amino Acid Sequence↗

Heat-induced degradation of PER and TIM in Drosophila bearing a conditional allele of the heat shock transcription factor gene.

Heat pulses elicit dramatic and rapid decreases in the levels of the D. melanogaster period (per) and timeless (tim) proteins (i.e., PER and TIM). To investigate the possible role of the heat shock pathway in this response, we used Drosophila bearing a conditional allele of the hsf gene (termed hsf4), which encodes the heat shock transcription factor (HSF). At all times in a daily cycle, heat-induced decreases in the levels of PER and TIM were similar in wild-type and hsf4 mutant flies. The results strongly suggest that the heat shock pathway contributes little, if any, to the response of the Drosophila circadian clock to heat signals.

Alleles↗

Induction of heat shock protein 72 in RGCs of rat acute glaucoma model after heat stress or zinc administration.

PURPOSE: To investigate the dynamics of heat shock protein 72 (HSP72) expression in retinal ganglion cells (RGCs) in rat model of acute glaucoma treated with heat stress or intraperitoneal injection of zinc sulfate. METHODS: Twenty-seven male Wistar rats were used to make acute glaucoma models. Five others served as normal control. Acute glaucoma models were made by intracameral irrigation in the right eyes with balanced salt saline (BSS) at 102 mmHg for 2 hours. Nine model rats were killed at different intervals after intracameral irrigation without treatment, which served as damage control. Ten were treated with heat stress 40 degrees C-42 degrees C, and 8 were used for zinc sulfate administration 2 days posterior to intracameral irrigation. Treated model rats were sacrificed at designed intervals after treatment. Right eyes were enucleated immediately, and the retinas were dissected for Western blot. RESULTS: No HSP72 was found in RGCs of normal Wistar rats. In damage control group, slight HSP72 was detected during 6-36 hours posterior to intracameral irrigation. HSP72 was detected significantly expressed in RGCs of both heat shock group and zinc sulfate group. But the dynamics of HSP72 production were quite different in these two treated groups. In heat shock group, HSP72 appeared at the sixth hour after treatment, and increased gradually until its peak production emerged at the 48th hour. HSP72 vanished 8 days later after treatment. In zinc sulfate group, HSP72 expression began 24 hours later after zinc administration, and reached its highest level at the 72th hour posterior to treatment. HSP72 expression then decreased slowly, and disappeared 21 days later after treatment. CONCLUSION: HSP72 can be induced in RGCs of rat acute glaucoma models with heat stress or zinc sulfate administration. But the dynamics of the HSP72 induction in those two groups were quite different.

Animals↗

Enhancement of hydroxyapatite-mediated three-dimensional-like proliferation of mouse fibroblasts by heat treatment: effects of heat shock-induced p38 MAPK pathway.

Regulation of the biocompatibility of compositional hydroxyapatite (HA) with cells is affected by various environmental factors. The aim of this study was to determine whether the p38 mitogen-activated protein kinase (MAPK) pathway has a key role in enhancement of HA-mediated three-dimensional (3D)-like proliferation of mouse fibroblasts after heat treatment. C3H10T1/2 mouse fibroblasts were cultured with HA granules for 10 weeks after heat treatment at 44 degrees C for 5, 10, 20, and 30 min. The mean rate of formation of 3D-like proliferation patterns by cells heat treated for 20 min was only 2.1-fold higher than that by untreated cells, but the mean rates of formation of 3D-like proliferation patterns by cells heat treated for 5 and 10 min were significantly higher (3.7- and 3.3-fold higher, respectively) than that by untreated cells (p < 0.01). Western blot analysis demonstrated that phosphorylation of p38 MAPK was markedly increased by heat treatment at 44 degrees C for 5 and 10 min. In addition, the activation of heat shock-induced p38 MAPK was markedly reduced by treatment at 44 degrees C for 30 min. We concluded that 3D-like proliferation of heat-treated cells was induced by activation of p38 MAPK. The results of this study should be useful for further studies aimed at elucidation of regulation of the biocompatibility of compositional HA with cells.

Animals↗

Proteomic analysis of the effect of heat stress on hexaploid wheat grain: characterization of heat-responsive proteins from non-prolamins fraction.

The effect of heat stress on hexaploid wheat grain proteome was recently analyzed in our previous works. Proteomic tools allowed the characterization of heat-responsive proteins of total endosperm, composed mainly of prolamins. The present work completes this study; our aim was to analyze the effect of heat stress on the water-soluble fraction, composed essentially of albumins and globulins. These proteins were separated by two-dimensional electrophoresis (2-DE), visualized by Coomassie Brilliant Blue (CBB) staining and analyzed by Melanie-3 software. Of the 43 heat-changed proteins, 24 were found to be up-regulated whereas 19 spot proteins were down-regulated. All of these proteins were subjected to matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by database searching which allowed the identification of 42 spots. Of these, some were enzymes involved in different metabolic pathways of plants, such as granule-bound starch synthase and glucose-1-phosphate adenyltransferase, involved in the starch synthesis pathway; beta-amylase, involved in carbohydrate metabolism, and the ATP synthase beta-chain that was related to four heat-decreased proteins. Moreover, five heat up-regulated proteins showed similarities with small heat shock proteins while three other spots were related to elongation factors or eucaryotic translation initiation factors. Proteins involved in abiotic stresses or in plant defense mechanism were also identified and are discussed.

Databases, Protein↗

Effects of 6 versus 12 days of heat acclimation on heat tolerance in lightly exercising men wearing protective clothing.

This study investigated the influence of 6 versus 12 days of heat acclimation on the tolerance of low-intensity exercise in the heat while wearing protective clothing. Sixteen young men were acclimated by treadmill walking (50% of each subject's maximal aerobic power for 60 min.day-1) in a climatic chamber [40 degrees C dry bulb (db), 30% relative humidity] for either 6 consecutive days or two 6-day periods, separated by a 1-day rest. Before and after heat acclimation, the subjects performed a heat-exercise test (1.34 m.s-1, 0% grade; 40 degrees C db, 30% relative humidity), either under control conditions [wearing normal light combat clothing (continuous exercise; n = 5)] or when wearing protective clothing resistant against nuclear, biological, and chemical (NBC) agents (repeated bouts of 15-min walk + 15-min rest; n = 8). Criteria for halting the test exercise were a rectal temperature (Tre) of 39.3 degrees C, a heart rate (fc) > or = 95% of the subject's observed maximum, unwillingness of the subject to continue, or the elapse of 150 min. Heat acclimation decreased overall test values of Tre, fc, and mean skin temperature for both control and protective clothing conditions. When wearing normal combat clothing, acclimation responses were about twice as large after 12 than after 6 days, but the response was not increased by longer acclimation when wearing NBC protective clothing. Both 6 and 12 days of acclimation increased tolerance times in NBC protective clothing by about 15 min [from 97 (4) to 112 (6) min and from 108 (10) to 120 (10) min for 6 and 12 days, respectively]. We conclude that the physiological strain and limitation of heat-exercise tolerance imposed by wearing NBC protective clothing are not reduced if heat acclimation is prolonged from 6 to 12 days.

Acclimatization↗

Tetracaine modifies the fragmentation mode of heated human erythrocytes and can induce heated cell fusion.

It is known that human erythrocytes in saline fragment by development of an unstable surface wave on the cell rim when cells are heated through the denaturation temperature of the structural protein, spectrin. Here the influence of tetracaine on the fragmentation process has been recorded and analysed by video microscopy of cells heated in rectangular glass microcapillaries. The number of waves per cell rim decreases with increasing tetracaine concentration until, at 0.5 mM tetracaine, wave growth on the cell rim is suppressed on most cells and the cells internalize membrane at the cell dimple. The rate constant for the change in the number of waves per cell with increasing tetracaine concentration is 9.6 mM-1 at a heating rate of 0.5 K/s. 50% of heated cells internalize membrane at 0.14 mM tetracaine. When cells are heated rapidly in suspension in test tubes the presence of tetracaine reduces the temperature for 50% haemolysis from 66 degrees C for washed control cells to 60.5 degrees C for cells in 2 mMs tetracaine. Cells heat in microcapillaries in tetracaine concentrations of 3 mM and higher begin to swell before the spectrin denaturation temperature is reached. Cell fusion was observed at and above the spectrin denaturation temperature in cells heated in 3 and 4 mM tetracaine. It was also noted that the morphology of erythrocytes maintained in 3.6 mM tetracaine for times up to 30 min at 37 degrees C or 20 degrees C was strongly dependent on temperature and time.

Cell Fusion↗

In vitro translation of Drosophila heat-shock and non--heat-shock mRNAs in heterologous and homologous cell-free systems.

Upon heat shock, Drosophila Kc cells still contain normal cellular messenger RNAs in the cytoplasm. The distribution of these 25 degrees C mRNAs between polysomes and the postpolysomal fraction of heat-shocked cells appears unaltered as compared with control cells. The translatability of these normal cellular messages isolated from heat-shocked and non--heat-shocked Kc cells is unaltered when analyzed by in vitro translation in the rabbit reticulocyte lysate. In contrast, homologous cell-free translation systems obtained from Kc cells effectively discriminate between the in vitro translation of normal cellular messages and heat-shock--specific mRNAs. In particular, a cell-free system from heat-shocked Drosophila Kc cells almost completely shuts down the translation of 25 degrees C messenger RNA species, whereas the translatability of heat-shock--specific messenger RNA appears to be unaffected.

Animals↗

A diffusion model for drying of a heat sensitive solid under multiple heat input modes.

To obtain optimal drying kinetics as well as quality of the dried product in a batch dryer, the energy required may be supplied by combining different modes of heat transfer. In this work, using potato slice as a model heat sensitive drying object, experimental studies were conducted using a batch heat pump dryer designed to permit simultaneous application of conduction and radiation heat. Four heat input schemes were compared: pure convection, radiation-coupled convection, conduction-coupled convection and radiation-conduction-coupled convection. A two-dimensional drying model was developed assuming the drying rate to be controlled by liquid water diffusion. Both drying rates and temperatures within the slab during drying under all these four heat input schemes showed good accord with measurements. Radiation-coupled convection is the recommended heat transfer scheme from the viewpoint of high drying rate and low energy consumption.

Convection↗

Heat-induced cobalt entry: an assay for heat transduction in cultured rat dorsal root ganglion neurons.

A histochemical stain to detect cobalt in cells was used to investigate the ionic basis of heat transduction in mammalian primary afferent neurons. Cultured dorsal root ganglion neurons from the adult rat were exposed to 10-min heat stimuli in an extracellular solution containing cobalt ions. When accumulated intracellular cobalt was precipitated, a subpopulation of neurons was darkly stained. The number of neurons stained depended on the intensity of the heat stimulus, ranging from 1.9% at 22 degrees C to 24.0% at 45 degrees C, a range of temperatures transduced by primary afferent nerve endings in vivo. Results of Trypan Blue exclusion experiments demonstrate that the heat-induced stain is not due to membrane damage, suggesting that heat opens a divalent-permeable ion channel. Agents that block many multivalent cation-permeable channels (lanthanum, ruthenium red and amiloride) did not reduce the number of cells that exhibited heat-induced cobalt staining. Heat-evoked cobalt staining provides an in vitro model for the investigation of the ionic mechanisms of thermal transduction in sensory neurons.

Animals↗

Characterization of the heat treatment undergone by milk using two inhibition ELISAs for quantification of native and heat denatured alpha-lactalbumin.

Dairy industries are interested to know the heat treatment undergone by milk for controlling the quality of drinking milks or to control their heating systems. The purpose of this work was to develop a specific and sensitive technique for classification of the heat treatment a milk has been submitted to, without disposing of the original raw milk. For this purpose, alpha-lactalbumin was chosen as a bioindicator of heat treatment, and monoclonal antibodies specific for its native or heat-denatured form were raised and used in two inhibition ELISAs. ELISA allowed differentiation among raw, pasteurized, ultrahigh-temperature-treated, and sterilized milks without even having to know the alpha-lactalbumin concentration of the original raw milk. However, this technique was more suitable for intense heat treatments such as UHT treatment and sterilization because of the heat stability of alpha-lactalbumin.

Animals↗