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PubMed1986

DNA polymerase activity in heat killing and hyperthermic radiosensitization of mammalian cells as observed after fractionated heat treatments.

Possible relations between hyperthermic inactivation of alpha and beta DNA polymerase activity and hyperthermic cell killing or hyperthermic radiosensitization were investigated. Ehrlich Ascites Tumor (EAT) cells and HeLa S3 cells were treated with fractionated doses of hyperthermia. The heating schedules were chosen such that the initial heat treatment resulted in either thermotolerance or thermosensitization (step-down heating) for the second heat treatment. The results show that for DNA polymerase activity and heat radiosensitization (cell survival) no thermotolerance or thermosensitization is observed. Thus hyperthermic cell killing and DNA polymerase activity are not correlated. The correlation of hyperthermic radiosensitization and DNA polymerase activity was substantially less than observed in previous experiments with normotolerant and thermotolerant HeLa S3 cells. We conclude that alpha and beta DNA polymerase inactivation is not always the critical cellular process responsible for hyperthermic cell killing or hyperthermic radiosensitization. Other possible cellular systems that might determine these processes are discussed.

Animals↗
PubMed1979

Relationships among training, heat acclimation, and heat tolerance in men and women: the controversy revisited.

For more than a decade there has been a controversy over the beneficial effects of physical training in a cool environment on exercise-heat tolerance. The issues include (a) inadequate controls regarding the physical characteristics of subjects, (b) marked variability in the conditions of the standard heat tolerance tests and (c) differences in the intensity and duration of the training programs employed. Intense training in a cool environment cannot serve as a substitute for exercise in the heat if acclimation is desired within a 2 week period. However, a substantial improvement (50%) in heat tolerance can be derived from 8-11 weeks of training under temperate conditions (21 degrees C) and thermal equilibrium can be maintained for at least 4 hours during mild work (200 W/m2) in dry or wet heat by endurance runners. These adjustments occur in both men and women and appear to be independent of aerobic capacity. The key to improved thermal tolerance with training in a cool environment is maintenance of an elevated core temperature for a sufficient duration of time to produce an adaptive response.

Acclimatization↗
PubMed1983

Muscarinic receptors in porcine caudate nucleus. II. Different effects of N-ethylmaleimide on [3H]cis-methyldioxolane binding to heat-labile (guanyl nucleotide-sensitive) sites and heat-stable (guanyl nucleotide-insensitive) sites.

Heat treatment of membranes from porcine caudate nucleus (50 degrees for 7 min) caused a marked decrease in [3H]cis-methyldioxolane [( 3H]CD) binding without affecting seriously the binding of [3H]3-quinuclidinyl benzilate [( 3H]QNB). Approximately 20% of the [3H]CD binding at 5 nM [3H]CD remained after the heat treatment. The remaining binding was not affected by 0.1 mM guanylyl-5'-imidodiphosphate (GppNHp) or by nickel or other cations at concentrations below 10 mM. Treatment of the membranes with trypsin (30 micrograms/mg of protein) at 20 degrees for 20 min also caused a marked decrease in [3H]CD binding without affecting seriously the binding of [3H]QNB. About 20% of the original [3H]CD binding remained in the presence of trypsin at a high concentration of protein (90 micrograms/mg). N-Ethylmaleimide (NEM) affected [3H]CD binding in two different ways: (a) preincubation of the membranes with NEM caused a marked reduction in heat- and GppNHp-sensitive [3H]CD binding, and (b) treatment with NEM caused an enhancement of heat-, GppNHp-, and trypsin-insensitive [3H]CD binding. Neither of the NEM effects required the coexistence of agonists. The concentration of NEM required for the first effect was 10 times lower than that for the second effect, indicating the existence of two NEM-binding sites with different affinities for NEM. The equilibrium dissociation constant (Kd) for [3H]CD after NEM treatment was 33 nM and was not affected by GppNHp, Ni2+, or heat treatment; the Kd was only 4 times higher than that (8 nM) without NEM treatment. These findings indicated the existence of two kinds of [3H]CD binding sites with high affinities for agonists: one is sensitive to guanyl nucleotide and is abolished by NEM and the other is induced by NEM and insensitive to guanyl nucleotide.

Animals↗
PubMed1981

Effect of microwave heating in cook/chill food service system. Energy balance for heating beef loaf.

In the microwave-heating subsystem of the simulated cook/chill food service, 100-gm. portions of precooked beef loaf were heated 80 sec. (2,450 mHz; 237 V.; 3,003 W. input power; 1,407 W. output power) to a mean internal end temperature of 82 +/- 9 degrees C. Mean input power to the microwave oven during heating, controlled by a transformer, was 240 kJ. per 80 sec. Fifty-three percent of the input power to the microwave oven was used to operate the oven. Of the 47 percent output power available to heat the beef loaf, 12 percent of the total input power was estimated to heat beef loaf, 17 percent was estimated to evaporate water from beef loaf, and 18 percent was attributed to miscellaneous energy losses.

Animals↗
PubMed1993

A critical role for heat shock transcription factor in establishing a nucleosome-free region over the TATA-initiation site of the yeast HSP82 heat shock gene.

Heat shock genes are poised for rapid transcriptional activation in response to environmental stress. A universal structural characteristic of such genes is the presence of a nucleosome-free, DNase I hypersensitive promoter region. Here we investigate the structural and functional effects of mutating HSE1, the preferred heat shock factor (HSF) binding site upstream of the yeast HSP82 gene. In situ deletion or substitution of this sequence reduces both basal and induced transcription by at least two orders of magnitude. Moreover, such mutations lead to a dramatic transition in chromatin structure: the DNase I hypersensitive region is replaced by two stable, sequence-positioned nucleosomes. One of these is centered over the mutated heat shock element, while the other--as revealed by DNase I genomic footprinting--is precisely positioned in a rotational sense over the TATA-initiation site. Overexpression of yeast HSF strongly suppresses the null phenotype of the induced hsp82-delta HSE1 gene and re-establishes DNase I hypersensitivity over its promoter. Such suppression is mediated through sequence disposed immediately upstream of HSE1 and containing two low affinity heat shock elements. These data imply a critical role for HSF in displacing stably positioned nucleosomes in Saccharomyces cerevisiae and suggest that HSF transcriptionally activates HSP82 at least partly through its ability to alleviate nucleosome repression of the core promoter.

Base Sequence↗
PubMed1995

Heat shock factor-1 and the heat shock cognate 70 protein associate in high molecular weight complexes in the cytoplasm of NIH-3T3 cells.

Interaction of heat shock transcription factor-1 (HSF-1) with the seventy kilodalton heat shock cognate protein (HSC70) was examined in NIH 3T3 cells. HSF-1 was found in the cytoplasm of non-stressed cells associated with HSC70 in large (Mr 400-500,000) complexes. After heat shock, HSF-1 became concentrated in the nucleus in smaller, more stable complexes that did not contain HSC70, an indication of significant rearrangement within the complexes. These experiments show a profound effect of heat shock on the structure and stability of HSF-1 complexes during nuclear localization and support the hypothesis that HSC70 binding may control HSF-1 function.

3T3 Cells↗
PubMed2001

Transcription of the ibpB heat-shock gene is under control of sigma(32)- and sigma(54)-promoters, a third regulon of heat-shock response.

The expression of the ibpAibpB heat-shock operon of Escherichia coli was found previously not to conform to the known pattern of expression of the sigma(32)-regulated operons because the rpoH gene mutation inactivating the sigma(32) protein did not abolish the ibp induction. We show here that this effect can depend partly on the sigma(54)-promoter that is inducible by heat shock, located upstream of the ibpB, the distal gene of the operon. It may also depend on a metabolic signal, postulated by others, and possibly required for the expression of the ibpAB genes. Thus, the ibpB gene can be translated from the transcript covering the whole operon starting from the sigma(32)-promoter and from the ibpB gene transcript starting from the sigma(54)-promoter. These results indicate that the ibpB gene is a second member of the sigma(54)-heat-shock regulon in E. coli besides pspA-E operon. Thus, heat-shock response involves three regulons controlled by sigma(32), sigma(24), and sigma(54) RNA polymerase subunits.

Bacterial Proteins↗
PubMed1995

Analysis by confocal microscopy of the behavior of heat shock protein 70 within the nucleus and of a nuclear matrix polypeptide during prolonged heat shock response in HeLa cells.

By means of confocal laser scanning microscopy and indirect fluorescence experiments we have examined the behavior of heat-shock protein 70 (HSP70) within the nucleus as well as of a nuclear matrix protein (M(r) = 125 kDa) during a prolonged heat-shock response (up to 24 h at 42 degrees C) in HeLa cells. In control cells HSP70 was mainly located in the cytoplasm. The protein translocated within the nucleus upon cell exposure to hyperthermia. The fluorescent pattern revealed by monoclonal antibody to HSP70 exhibited several changes during the 24-h-long incubation. The nuclear matrix protein showed changes in its location that were evident as early as 1 h after initiation of heat shock. After 7 h of treatment, the protein regained its original distribution. However, in the late stages of the hyperthermic treatment (17-24 h) the fluorescent pattern due to 125-kDa protein changed again and its original distribution was never observed again. These results show that HSP70 changes its localization within the nucleus conceivably because it is involved in solubilizing aggregated polypeptides present in different nuclear regions. Our data also strengthen the contention that proteins of the insoluble nucleoskeleton are involved in nuclear structure changes that occur during heat-shock response.

Antibodies, Monoclonal↗
PubMed2006

Protective role of heat shock and heat shock protein 70 in lactacystin-induced cell death both in the rat substantia nigra and PC12 cells.

Proteasomal dysfunction plays an important role in the pathogenesis of Parkinson disease (PD). Although clinical and experimental evidence continues to accumulate indicating heat shock protein 70 (HSP70) is significant in the pathogenesis of PD, few studies have been made to investigate the role of HSP70 under the condition of proteasome dysfunction. In in vivo study, we infused lactacystin into the unilateral substantia nigra (SN) of Sprague-Dawley rats with or without preceding whole body hyperthermia (WBH). Immunohistochemical studies showed the death of dopaminergic neurons and activated microglia in the SN. Lactacystin with prior WBH increased the expression of HSP70 more than did lactacystin alone and decreased lactacystin-induced dopaminergic neuronal death in the SN. In PC12 cells, heat shock pretreatment decreased lactacystin-induced cell death. Although additional treatment of nocodazole, ammonium chloride, and 3-methyladenine augmented cell death by lactacystin, heat shock pretreated to these drugs offsets their additional toxicity. These results indicate that heat shock proteins, especially HSP70, could play an important role under the condition of proteasome dysfunction in part by fostering aggresome formation and lysosome-mediated autophagy.

Acetylcysteine↗
PubMed2006

Molecular cloning of the heat-shock cognate 70 (Hsc70) gene from the two-spotted spider mite, Tetranychus urticae, and its expression in response to heat shock and starvation.

We isolated a heat shock cognate 70 (hsc70) gene from the two-spotted spider mite, Tetranychus urticae, a serious agricultural pest. The hsc70 cDNA is 2275 bp and contains a 1962 bp open reading frame. The translated amino acid sequence consists of 654 residues with a calculated molecular mass of 71,275 Da and an isoelectronic point (pI) of 5.52. It also contains the highly conserved functional motifs of the Hsp70 family. A comparison of the deduced amino acid sequence shows a high identity (81-84%) with Hsp70s/Hsc70s of insects but the highest identity is with mussel Hsc71 (86%). Northern blot hybridization indicates that the hsc70 transcript level of female adults is higher than that of male adults. We evaluated the response of hsc70 gene to stresses from temperature and starvation. The level of hsc70 mRNA was not significantly changed by heat and cold shocks nor by recovery after the shocks. However, the hsc70 mRNA level was decreased by food restriction of female mites. Analysis of nucleotide and deduced amino acid sequences of hsc70 gene from T. urticae suggests that it is a member of heat shock cognate 70 gene in the highly conserved Hsp70 family but that its expression is influenced by food restriction rather than thermal stress. This is the first molecular analysis of a heat shock protein gene in an acarid.

Amino Acid Sequence↗
PubMed2006

Extracellular heat shock protein 70 mediates heat stress-induced epidermal growth factor receptor transactivation in A431 carcinoma cells.

The initial steps of heat stress in A431 cells were previously characterized by ligand-independent EGFR transactivation via an unknown mechanism and concomitant secretion of Hsp70. In this work we demonstrate that the depletion of Hsp70 from the conditioned medium of heated cells abolishes EGFR transactivation indicating that secreted Hsp70 is essential for EGFR transactivation during heat shock. This notion is supported by the findings that purified Hsp70 can induce EGFR transactivation and the activation of EGFR-dependent signaling pathways. Both heat stress and pure Hsp70 stimulate activation of TLR2/4 and their association with EGFR. These results suggest that the secreted Hsp70 mediates the cross-communication of TLR and EGFR signaling systems in A431 cells.

Animals↗
PubMed2003

Identification of cerebral neurons and glial cell types inducing heat shock protein Hsp70 following heat stress in the rat.

Heat shock proteins were recently recognized as molecular chaperones that besides their chaperoning function were also involved in processes of cell death and survival. Many types of neural cells were reportedly capable of expressing heat shock protein Hsp70 following heat stress in vitro. However, identification of cell types inducing Hsp70 protein in the hyperthermic brain is not clear. In this study, cerebral Hsp70 distribution was evaluated in anesthetized adult rats (urethane, 1.5 g/kg, i.p.) subjected to short-term hyperthermia (41.5 degrees C for 30 min). Detection of Hsp70 was achieved by an ABC technique in vibratome or paraffin sections combined with specific markers of glial cell types. Hsp70 appeared by 90 min, mainly in glial and vascular cells, with enhanced immunostaining by 4 h following hyperthermia. Higher numbers of Hsp70-positive cells were detected in the white matter and diencephalic region than in the cerebral cortex, especially over the shorter interval. Hsp70 was localized in many oligodendrocytes, double-labeled with lectin GSII, and some vessels. Microglia showed apparently less Hsp70/OX-42 double-labeled cells than the previous two cell types. In contrast, only a few Hsp70-stained cells were positive for astrocyte marker GFAP. In addition to glial/vascular Hsp70 staining, neuronal Hsp70 induction was observed only in discrete regions including the paraventricular, supraoptic, suprachiasmatic and other hypothalamic nuclei, and in amygdala. Prevailing heat-stress expression of Hsp70 in oligodendrocytes and vascular cells might render them less susceptible to the consequences of other types of cell stress and could be exploited to increase selectively their survival in pathological situations.

Amygdala↗
PubMed2000

Heat shock-induced arrests in different cell cycle phases of rat C6-glioma cells are attenuated in heat shock-primed thermotolerant cells.

The response kinetics of rat C6 glioma cells to heat shock was investigated by means of flow cytometric DNA measurements and western blot analysis of HSP levels. The results showed that the effects on cell cycle progression are dependent on the cell cycle phase at which heat shock is applied, leading to either G1 or G2/M arrest in randomly proliferating cells. When synchronous cultures were stressed during G0 they were arrested with G1 DNA content and showed prolongation of S and G2 phases after release from the block. In proliferating cells, HSC70 and HSP68 were induced during the recovery and reached maximum levels just before cells were released from the cell cycle blocks. Hyperthermic pretreatment induced thermotolerance both in asynchronous and synchronous cultures as evidenced by the reduced arrest of cell cycle progression after the second heat shock. Thermotolerance development was independent of the cell cycle phase. Pre-treated cells already had high HSP levels and did not further increase the amount of HSP after the second treatment. However, as in unprimed cells, HSP reduction coincided with the release from the cell cycle blocks. These results imply that the cell cycle machinery can be rendered thermotolerant by heat shock pretreatment and supports the assumption that HSP70 family members might be involved in thermotolerance development.

Adaptation, Physiological↗
PubMed1999

Purification and characterization of the 16-kDa heat-shock-responsive protein from the thermophilic cyanobacterium Synechococcus vulcanus, which is an alpha-crystallin-related, small heat shock protein.

A 16-kDa protein, one of the major proteins that accumulates upon heat-shock treatment in the thermophilic cyanobacterium Synechococcus vulcanus, was purified to apparent homogeneity. The N-terminal and internal amino acid sequences of the protein exhibited a homology to the alpha-crystallin-related, small heat shock proteins from other organisms. The protein was designated HspA. Size-exclusion chromatography and nondenaturing gel electrophoresis demonstrated that HspA formed a large homo-oligomer consisting of 24 subunits. It prevented the aggregation of porcine malic dehydrogenase at 45 degrees C and 50 degrees C and citrate synthase at 50 degrees C. The activity of the malic dehydrogenase, however, was not protected under these heat-shock conditions or reactivated after a shift in temperature from 45 or 50 degrees C to 21 degrees C. HspA was able to enhance the refolding of chemically denatured rabbit muscle lactate dehydrogenase in an ATP-independent manner. A homologue to the 16-kDa protein was also found to be induced upon heat-shock treatment in the mesophilic cyanobacterium Synechocystis sp. PCC 6803.

Amino Acid Sequence↗
PubMed2003

The identity of proteins associated with a small heat shock protein during heat stress in vivo indicates that these chaperones protect a wide range of cellular functions.

The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has been studied extensively in vitro, understanding the mechanism of sHSP function requires identification of proteins that are sHSP substrates in vivo. We have used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6 in vivo during heat treatment. These proteins can all be released from Hsp16.6 by the ATP-dependent activity of DnaK and co-chaperones and are heat-labile. Thirteen of the putative substrate proteins were identified by mass spectrometry and reveal the potential for sHSPs to protect cellular functions as diverse as transcription, translation, cell signaling, and secondary metabolism. One of the putative substrates, serine esterase, was purified and tested directly for interaction with purified Hsp16.6. Hsp16.6 effectively formed soluble complexes with serine esterase in a heat-dependent fashion, thereby preventing formation of insoluble serine esterase aggregates. These data offer critical insights into the characteristics of native sHSP substrates and extend and provide in vivo support for the chaperone model of sHSP function.

Adenosine Triphosphate↗
PubMed2005

Immunomodulation of function of small heat shock proteins prevents their assembly into heat stress granules and results in cell death at sublethal temperatures.

The conformational dynamism and aggregate state of small heat shock proteins (sHSPs) may be crucial for their functions in thermoprotection of plant cells from the detrimental effects of heat stress. Ectopic expression of single chain fragment variable (scFv) antibodies against cytosolic sHSPs was used as new tool to generate sHSP loss-of-function mutants by antibody-mediated prevention of the sHSP assembly in vivo. Anti-sHSP scFv antibodies transiently expressed in heat-stressed tobacco protoplasts were not only able to recognize the endogenous sHSPs but also prevented their assembly into heat stress granula (HSGs). Constitutive expression of the same scFv antibodies in transgenic plants did not alter their phenotype at normal growth temperatures, but their leaves turned yellow and died after prolonged stress at sublethal temperatures. Structural analysis revealed a regular cytosolic distribution of stress-induced sHSPs in mesophyll cells of stress-treated transgenic plants, whereas extensive formation of HSGs was observed in control cells. After prolonged stress at sublethal temperatures, mesophyll cells of transgenic plants suffered destruction of all cellular membranes and finally underwent cell death. In contrast, mesophyll cells of the stressed controls showed HSG disintegration accompanied by appearance of polysomes, dictyosomes and rough endoplasmic reticulum indicating normalization of cell functions. Apparently, the ability of sHSPs to assemble into HSGs as well as the HSG disintegration is a prerequisite for survival of plant cells under continuous stress conditions at sublethal temperatures.

Cell Death↗
PubMed2005

Saccharomyces cerevisiae heat shock transcription factor regulates cell wall remodeling in response to heat shock.

The heat shock transcription factor Hsf1 of the yeast Saccharomyces cerevisiae regulates expression of genes encoding heat shock proteins and a variety of other proteins as well. To better understand the cellular roles of Hsf1, we screened multicopy suppressor genes of a temperature-sensitive hsf1 mutation. The RIM15 gene, encoding a protein kinase that is negatively regulated by the cyclic AMP-dependent protein kinase, was identified as a suppressor, but Rim15-regulated stress-responsive transcription factors, such as Msn2, Msn4, and Gis1, were unable to rescue the temperature-sensitive growth phenotype of the hsf1 mutant. Another class of suppressors encoded cell wall stress sensors, Wsc1, Wsc2, and Mid2, and the GDP/GTP exchange factor Rom2 that interacts with these cell wall sensors. Activation of a protein kinase, Pkc1, which is induced by these cell wall sensor proteins upon heat shock, but not activation of the Pkc1-regulated mitogen-activated protein kinase cascade, was necessary for the hsf1 suppression. Like Wsc-Pkc1 pathway mutants, hsf1 cells exhibited an osmotic remedial cell lysis phenotype at elevated temperatures. Several of the other suppressors were found to encode proteins functioning in cell wall organization. These results suggest that Hsf1 in concert with Pkc1 regulates cell wall remodeling in response to heat shock.

Calcium-Binding Proteins↗
PubMed1993

Activation of heat shock gene transcription by heat shock factor 1 involves oligomerization, acquisition of DNA-binding activity, and nuclear localization and can occur in the absence of stress.

The existence of multiple heat shock factor (HSF) genes in higher eukaryotes has promoted questions regarding the functions of these HSF family members, especially with respect to the stress response. To address these questions, we have used polyclonal antisera raised against mouse HSF1 and HSF2 to examine the biochemical, physical, and functional properties of these two factors in unstressed and heat-shocked mouse and human cells. We have identified HSF1 as the mediator of stress-induced heat shock gene transcription. HSF1 displays stress-induced DNA-binding activity, oligomerization, and nuclear localization, while HSF2 does not. Also, HSF1 undergoes phosphorylation in cells exposed to heat or cadmium sulfate but not in cells treated with the amino acid analog L-azetidine-2-carboxylic acid, indicating that phosphorylation of HSF1 is not essential for its activation. Interestingly, HSF1 and HSF2 overexpressed in transfected 3T3 cells both display constitutive DNA-binding activity, oligomerization, and transcriptional activity. These results demonstrate that HSF1 can be activated in the absence of physiological stress and also provide support for a model of regulation of HSF1 and HSF2 activity by a titratable negative regulatory factor.

3T3 Cells↗