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Heat illness. Fluid and electrolyte issues for pediatric and adolescent athletes.

The primary mechanism for maintaining normal body temperature during physical exercise in the heat is the evaporation of sweat. With profuse sweating, water loss far exceeds electrolyte loss. Rigorous exercise in the heat places the athlete at risk for thermoregulatory dysfunction from dehydration. Because children are inherently less efficient thermoregulators than adults, they are at even greater risk for heat illness. The three primary syndromes of heat illness are heat cramps, heat exhaustion, and heat stroke. Treatment of heat illness is based on reduction of body temperature and rehydration. Heat stroke is a true medical emergency with a high mortality rate; immediate reduction of body temperature is critical to the survival of these patients. Prevention of heat illness is based on reducing known risk factors. Physical activity should be modified in the face of high ambient temperature and humidity. The athlete should begin exercise well hydrated; frequent consumption of cold water during exercise decreases likelihood of significant dehydration. After exercise, the athlete should continue drinking to replace fluid losses. Clothing should be lightweight; the more skin exposed, the greater the available evaporative surface. A preseason conditioning program, when combined with an 8- to 14-day period of acclimatization, further reduces the risk of heat injury. Although athletes engaged in endurance sports may benefit from drinking carbohydrate/electrolyte-containing solutions, for the majority of young athletes, cold water remains the preferred choice for fluid replacement during exercise. The relatively greater body surface area of young athletes also places them at risk for hypothermia. Special attention should be given when these athletes are competing under cold environmental conditions.

Adolescent↗

The effect of culture growth phase on induction of the heat shock response in Yersinia enterocolitica and Listeria monocytogenes.

The effect of culture growth phase on induction of the heat shock response in Yersinia enterocolitica and Listeria monocytogenes, was examined. Exponential or stationary preconditioned cultures were heat shocked and survivor numbers estimated using selective and overlay/resuscitation recovery techniques. The results indicate that prior heat shock induced increased heat resistance in both micro-organisms to higher heat treatments. Heat-shocked cells of each micro-organism were able to survive much longer than non-heat-shocked cells when heated at 55 degrees C. The size of the change in heat resistance between heat-shocked and non-heat-shocked cells was greatest for exponential cultures (X:X). Results indicate that the overall relative thermal resistance of each pathogen was dependent on cell growth phase. Stationary cultures (S:S) were significantly (P < 0.01) more thermotolerant than exponential cultures (X:X) under identical processing conditions. Under most conditions, the use of an overlay/resuscitation recovery medium resulted in higher D-values (P < 0.05) compared with a selective recovery medium.

Colony Count, Microbial↗

Heat shock suppresses the permeability transition in rat liver mitochondria.

Heat shock proteins inhibit apoptotic and necrotic cell death in various cell types. However, the specific mechanism underlying protection by heat shock proteins remains unclear. To test the hypothesis that heat shock proteins inhibit cell death by blocking opening of mitochondrial permeability transition (MPT) pores, mitochondria from heat-preconditioned rat livers were isolated by differential centrifugation. Heat shock inhibited MPT pore opening induced by 50 microm CaCl(2) plus 5 microm HgCl(2) or 1 microm mastoparan and by 200 microm CaCl(2) alone. Half-maximal swelling was delayed 15 min or more after heat shock compared with control. Heat shock also increased the threshold of unregulated (Ca(2+)-independent and cyclosporin A-insensitive) MPT pore opening induced by higher doses of HgCl(2) and mastoparan. Heat shock treatment decreased mitochondrial reactive oxygen species formation by 27% but did not change mitochondrial respiration, membrane potential, Ca(2+) uptake, or total glutathione in mitochondrial and cytosolic extracts of liver. Western blot analysis showed that mitochondrial Hsp25 increased, whereas Hsp10, Hsp60, Hsp70, Hsp75, cyclophilin D, and voltage-dependent anion channel did not change after heat shock. These results indicate that heat shock causes resistance to opening of MPT pores, which may contribute to heat shock protection against cellular injury.

Animals↗

Heat stress and strain in an aluminum smelter.

Studies of worker heat stress and strain in aluminum smelters have found that heat exposure likely to exceed the American Conference of Governmental Industrial Hygienists' threshold limit value (TLV) and that the dose-response relationship between heat stress and strain was weak. A heat stress model based on climatic data and a task analysis indicated exposures to heat stress in excess of the TLV during the July/August study period. To study the impact of working above the TLV, heat strain data (i.e., oral temperature, recovery heart rate, average heart rate) were collected. Recovery heart rates indicated high strain most of the time, and oral temperatures after peak demands were above the no-strain threshold of 37.5 degrees C about a quarter of the time, indicating that heat stress had an effect. About 95% of the readings were below 38.0 degrees C, the acute oral temperature threshold for a safe exposure. Average heart rates over 6- and 12-hour intervals were generally below acceptable limits of 120 and 110 bpm, respectively. Oral temperature and average heart rates indicated good control of heat stress exposures. Because recovery heart rates were high, some employees were working near their individual limits. The dose-response relationship for recovery heart rate and oral temperature were examined against the level of heat stress above the TLV. There was no relationship between oral temperature and heat stress level. There was an apparent trend toward higher recovery heart rates with heat stress. The lack of a dose-response relationship may be explained by brief periods of very high wet bulb globe temperatures that drove the time-weighted average up out of proportion to the physiological response.

Aluminum↗

Fluid and electrolyte supplementation for exercise heat stress.

During exercise in the heat, sweat output often exceeds water intake, resulting in a body water deficit (hypohydration) and electrolyte losses. Because daily water losses can be substantial, persons need to emphasize drinking during exercise as well as at meals. For persons consuming a normal diet, electrolyte supplementation is not warranted except perhaps during the first few days of heat exposure. Aerobic exercise is likely to be adversely affected by heat stress and hypohydration; the warmer the climate the greater the potential for performance decrements. Hypohydration increases heat storage and reduces a person's ability to tolerate heat strain. The increased heat storage is mediated by a lower sweating rate (evaporative heat loss) and reduced skin blood flow (dry heat loss) for a given core temperature. Heat-acclimated persons need to pay particular attention to fluid replacement because heat acclimation increases sweat losses, and hypohydration negates the thermoregulatory advantages conferred by acclimation. It has been suggested that hyperhydration (increased total body water) may reduce physiologic strain during exercise heat stress, but data supporting that notion are not robust. Research is recommended for 3 populations with fluid and electrolyte balance problems: older adults, cystic fibrosis patients, and persons with spinal cord injuries.

Adult↗

Meiotic induction by heat stress in mouse oocytes: involvement of AMP-activated protein kinase and MAPK family members.

In this study, we examined the effect of heat pulsing on oocyte maturation and assessed the possible role of stress-activated enzymes during heat stress-induced meiotic maturation. Denuded oocytes from immature eCG-primed mice were pulsed for 30 min at increasing temperatures from 40 degrees C to 43 degrees C in dibutyryl cAMP-containing medium and were subsequently cultured at 37 degrees C for a total incubation time of 17-18 h. Oocytes exposed to 42 degrees C showed the greatest stimulation of maturation, with no effect at 43 degrees C. A heat pulse did not compromise progression to metaphase II as observed by polar body (PB) formation. The AMP-activated protein kinase (PRKA) inhibitors compound C and Ara-A each blocked the meiosis-stimulating effects of heat. Western blots showed that acetyl-CoA carboxylase, an important substrate of PRKA, was phosphorylated in heat-treated germinal vesicle-stage oocytes, indicating activation of PRKA before maturation. The mitogen-activated protein 2 kinase (MAP2K1) inhibitor PD98059 also prevented heat-induced maturation, but this effect was unrelated to MAPK1/3 activation, which was not observed until after germinal vesicle breakdown (GVB). Phosphorylated MAPK14 was not detected in the oocyte under any experimental condition, and only high concentrations of the MAPK14 inhibitor SB203580 blocked heat-stimulated maturation, suggesting that MAPK14 is not involved in meiotic induction. MAPK8/9 was activated by heat, and the MAPK8/9 inhibitor SP600125, but not JUN N-terminal kinase I, blocked heat-induced maturation. Heat treatment transiently suppressed GVB and PB formation in spontaneously maturing oocytes by a mechanism that is apparently different from its meiosis-inducing action. Collectively, these data show that an acute heat pulse stimulates GVB in meiotically arrested oocytes and suggest that this effect is mediated through the activation of PRKA.

AMP-Activated Protein Kinases↗

Induction of the heat shock response prevents tissue injury during acute inflammation of the rat ileum.

OBJECTIVES: To determine if prior total body hyperthermia protected against subsequent acute ileitis induced by the cytotoxic lectin, ricin, in rats. The time course of heat shock mRNA and protein expression in the ileum was determined. The effects of heat stress on small intestinal mucosal integrity, arachidonic acid metabolism, and neutrophilic infiltrate were compared in heated and nonheated rats receiving vehicle or ricin intraluminally. The effect of hyperthermia on the circulating neutrophil superoxide production was also evaluated. DESIGN: Prospective, randomized, controlled trial. SETTING: University research laboratory. SUBJECTS: Forty-one adult, male Sprague-Dawley rats, weighing 150 to 250 g, and 32 adult, inbred, male Fisher 344 rats, weighing 175 to 250 g. INTERVENTIONS: Exposure to whole body hyperthermia and production of acute ileitis. Sprague-Dawley rats were divided randomly into four experimental groups: nonheated control group, heated control group, nonheated ricin group (1 mg/mL water, intraluminal), and heated ricin group. Sprague-Dawley rats in a separate study were assigned to seven groups based on the time of removal of the terminal ileum following hyperthermia: 0 min, or 1, 2, 4, 8, 12, and 24 hrs. Inbred Fisher 344 rats were allocated to the heated and nonheated groups for peripheral neutrophil superoxide generation studies. MEASUREMENTS AND MAIN RESULTS: Whole body hyperthermia to a rectal temperature of 41 degrees C to 42 degrees C for 15 to 20 mins: a) was associated with marked mucosal cytoprotection against subsequent ricin-induced ileitis (Injury grade [from 0 = normal to 5 = severe]: 0.4 +/- 0.1 vs. 2.5 +/- 0.2, p < .001); b) prevented the ricin-induced reduction in villus height to crypt depth ratio (2.4 +/- 0.1 vs. 1.9 +/- 0.1, p < .01); and c) significantly reduced the number of infiltrating neutrophils when compared with nonheated ricin-treated rats (11 +/- 2 vs. 32 +/- 3 neutrophils/high-power field, p < .001). The hyperthermia-induced peak increase in heat shock protein (HSP)-70 mRNA at 2 hrs preceded that of HSP 70i at 4 hrs. Heat shock significantly reduced the ricin-induced increase in both basal (8.0 +/- 1.9 vs. 33.0 +/- 8.1 pg of leukotriene B4/mg protein, p < .05) and ionophore-stimulated (16.0 +/- 4.9 vs. 80.0 +/- 15.5 pg of leukotriene B4/mg protein, p < .001) generation of ileal leukotriene B4, but did not alter the cyclooxygenase product, prostaglandin E2. Hyperthermia did not alter peripheral neutrophil superoxide production. CONCLUSIONS: This study assessed the effects of heat shock in the normal and acutely inflamed intestine. These data suggest that heat stress and increased expression of HSP 70i protect against acute intestinal inflammation. This protection is associated with significant reductions in ileal leukotriene B4 generation and neutrophilic infiltrate. Hyperthermia did not alter circulating neutrophil superoxide production. Thus, the mechanism of heat stress protection against acute ileitis may involve local intestinal inhibition of leukotriene B4 production and subsequent neutrophilic infiltration without altering the ability of systemic neutrophils to be activated.

Animals↗

Heat stress increases survival rates in lipopolysaccharide-stimulated rats.

OBJECTIVE: To examine the hypothesis that heat stress applied after the administration of bacterial endotoxin is protective. DESIGN: Prospective, randomized, laboratory study. SETTING: University research laboratory. SUBJECTS: One hundred eleven adult male Sprague-Dawley rats (weight range 250 to 400 g). INTERVENTIONS: Production of endotoxemia by the administration of a bacterial endotoxin and exposure to heat stress by heating animals in a neonatal incubator until their rectal temperatures reached 105.8 degrees F (41 degrees C). MEASUREMENTS AND MAIN RESULTS: The rats (n = 111) were anesthetized and were injected with 15 mg/kg of Escherichia coli endotoxin (lipopolysaccharide, LPS) intravenously to produce septic shock. Immediately thereafter, a set of 50 rats were randomly assigned to one of two treatment groups: a) LPS-treated (control); or b) LPS-treated and heated to 105.8 degrees F (41 degrees C). The animals were then observed for the development of fever, and survival rates were monitored for 72 hrs. In another set of 40 animals, the same experimental protocol was used to determine plasma cytokine concentrations in heated and nonheated groups. Blood samples were obtained at 0, 2, 4, or 6 hrs after LPS injection for tumor necrosis factor-alpha and interleukin (IL)-1 beta detection. In a third set of animals, the same experimental protocol was applied to nine animals for the detection of heat-shock proteins of 72-kilodalton molecular weight. LPS injection in the control group did not produce fever. Heat stress increased the abundance of heat-shock proteins of 72-kilodalton molecular weight in the rats' lungs (analysis of variance, p = .016). Twelve hours after the initiation of sepsis, the survival rates of the control group injected with LPS alone and the group heated to 105.8 degrees F (41 degrees C) were 48% and 80%, respectively (p = .039). The peak plasma IL-1 beta concentrations occurring at 2 hrs after LPS injection were significantly reduced in rats heated to 105.8 degrees F (41 degrees C) when compared with nonheated rats (p = .003). CONCLUSION: We conclude that heat stress applied after the initiation of endotoxemia can provide protection against an otherwise lethal stimulus and that the mechanism of protection may be related to the attenuation of plasma IL-1 beta concentrations.

Animals↗

Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid.

Plants, in common with all organisms, have evolved mechanisms to cope with the problems caused by high temperatures. We examined specifically the involvement of calcium, abscisic acid (ABA), ethylene, and salicylic acid (SA) in the protection against heat-induced oxidative damage in Arabidopsis. Heat caused increased thiobarbituric acid reactive substance levels (an indicator of oxidative damage to membranes) and reduced survival. Both effects required light and were reduced in plants that had acquired thermotolerance through a mild heat pretreatment. Calcium channel blockers and calmodulin inhibitors increased these effects of heating and added calcium reversed them, implying that protection against heat-induced oxidative damage in Arabidopsis requires calcium and calmodulin. Similar to calcium, SA, 1-aminocyclopropane-1-carboxylic acid (a precursor to ethylene), and ABA added to plants protected them from heat-induced oxidative damage. In addition, the ethylene-insensitive mutant etr-1, the ABA-insensitive mutant abi-1, and a transgenic line expressing nahG (consequently inhibited in SA production) showed increased susceptibility to heat. These data suggest that protection against heat-induced oxidative damage in Arabidopsis also involves ethylene, ABA, and SA. Real time measurements of cytosolic calcium levels during heating in Arabidopsis detected no increases in response to heat per se, but showed transient elevations in response to recovery from heating. The magnitude of these calcium peaks was greater in thermotolerant plants, implying that these calcium signals might play a role in mediating the effects of acquired thermotolerance. Calcium channel blockers and calmodulin inhibitors added solely during the recovery phase suggest that this role for calcium is in protecting against oxidative damage specifically during/after recovery.

Abscisic Acid↗

The heat-shock response in Drosophila KC 161 cells. mRNA competition is the main explanation for reduction of normal protein synthesis.

The effect of heat shock on protein synthesis in the Drosophila melanogaster KC 161 tissue culture cell line was examined with a view to investigating the mechanism underlying the acute reduction in normal cellular protein synthesis typical of heat-shocked Drosophila cells. However, at 36-37 degrees C, the optimum temperature for induction of the 70-kDa heat-shock protein, this cell line did not show such a response. The synthesis of a very limited number of proteins was abruptly turned off following heat shock in the presence or absence of actinomycin, but the rate of synthesis of the majority of normal cellular proteins declined slowly over a three-hour period. Incubation of heat-shocked cells in hypertonic media increased the relative proportion of protein synthesis directed towards heat-shock proteins (as opposed to normal cellular proteins). Incubation with low concentrations of cycloheximide had the converse effect and resulted in a preferential increase in the size of polysomes translating normal cellular mRNAs, greater than the increase in size of polysomes synthesising heat-shock proteins. Heat shock also resulted in some mRNAs being almost completely displaced from polysomes into the postribosomal supernatant. These observations suggest that competition between normal cellular mRNAs and increasing amounts of heat-shock mRNAs with a higher affinity for the translation machinery was the main explanation for the gradual reduction in the synthesis of normal cellular proteins, although a slight reduction in overall translation initiation rates cannot be excluded as a subsidiary cause. The results demonstrate that the acute reduction in normal cellular protein synthesis seen in other Drosophila cell lines is not an integral and necessary feature of the heat-shock response in this organism, which makes it unlikely that the mechanism of this acute shut-off is intimately connected with the mechanism of induction of heat-shock mRNAs.

Animals↗

The nuclear-coded chloroplast 22-kDa heat-shock protein of Chlamydomonas. Evidence for translocation into the organelle without a processing step.

A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlamydomonas reinhardtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine alpha-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.

Amino Acid Sequence↗

Sheng mai san, a Chinese herbal medicine, protects against renal ischaemic injury during heat stroke in the rat.

1. There is evidence that the induction of inducible nitric oxide synthase (iNOS) and peroxynitrite by ischaemia/reperfusion may lead to renal cell injury. Herein, we investigated whether Sheng mai san (SMS), a Chinese herbal medicine, protects against renal ischaemic injury during heat stroke by reducing iNOS-dependent nitric oxide (NO) and peroxynitrite formation. 2. Urethane-anaesthetized rats were exposed to heat stress (ambient temperature 43 degrees C) to induce heat stroke. Control rats were exposed to 24 degrees C. Mean arterial pressure and renal blood flow after the onset of heat stroke were significantly lower in heat stroke rats than in control rats. However, both colonic temperature and renal damage score were greater in heat stroke rats compared with control rats. Similarly, plasma NO, creatinine and blood urea nitrogen (BUN), as well as the renal immunoreactivity of iNOS and peroxynitrite, were significantly higher in heat stroke rats compared with their normothermic controls. 3. Pretreatment with SMS (1.2 g/day per rat for 7 consecutive days before the initiation of heat stress) significantly attenuated the heat stroke-induced arterial hypotension, hyperthermia, renal ischaemia and damage, the increased renal immunoreactivity of iNOS and peroxynitrite and the increased plasma levels of NO, creatinine and BUN. Pretreatment with SMS resulted in a prolongation of survival time in heat stroke. 4. The results of the present study suggest that SMS protects against renal ischaemic damage by reducing iNOS-dependent NO and peroxynitrite production during heat stroke.

Animals↗

Changes in cell morphology and actin organization during heat shock in Dictyostelium discoideum: does HSP70 play a role in acquired thermotolerance?

In response to heat shock (34 degrees C, 30 min), cell morphology and actin organization in Dictyostelium discoideum are drastically changed. Loss of pseudopodia and disappearance of F-actin-containing structures were observed by using fluorescence microscopy. These changes were paralleled by a rapid decrease of the F-actin content measured by a TRITC-phalloidin binding assay. The effects of heat shock on cell morphology and actin organization are transient: After heat shock (34 degrees C) or during a long-term heat treatment (30 degrees C), cell morphology, F-actin patterns and F-actin content recovered/adapted to a state which is characteristic for untreated cells. Because F-actin may be stabilized by increased amounts of heat shock proteins, their response and interaction with F-actin was analyzed. After a 1 h heat treatment (34 degrees C), the major heat shock protein of D. discoideum (HSP70) showed maximally increased synthesis rates and levels. During recovery from a 34 degrees C shock or during a continuous heat treatment at 30 degrees C, the HSP70 content first increased and then declined slowly toward normal levels. Pre-treatment of cells with a short heat shock of 30 min at 34 degrees C stabilized the F-actin content when the cells were exposed to a second heat shock. Furthermore, a transient colocalization of HSP70 and actin was observed at the beginning of heat treatment (30 degrees C) using immunological detection of HSP70 in the cytoskeletal actin fraction.

Actins↗

The prolactin responses to active and passive heating in man.

The aim of this study was to compare the prolactin and blood pressure responses at identical core temperatures during active and passive heat stresses, using prolactin as an indirect marker of central fatigue. Twelve male subjects cycled to exhaustion at 60% maximal oxygen uptake (VO2peak) in a room maintained at 33 degrees C (active). In a second trial they were passively heated (passive) in a water bath (41.56 +/- 1.65 degrees C) until core temperature was equal to the core temperature observed at exhaustion during the active trial. Blood samples were taken from an indwelling venous cannula for the determination of serum prolactin during active heating and at corresponding core temperatures during passive heating. Core temperature was not significantly different between the two methods of heating and averaged 38.81 +/- 0.53 and 38.82 +/- 0.70 degrees C (data expressed as means +/- s.d.) at exhaustion during active heating and at the end of passive heating, respectively (P > 0.05). Mean arterial blood pressure was significantly lower throughout passive heating (active, 73 +/- 9 mmHg; passive, 62 +/- 12 mmHg; P < 0.01). Despite the significantly reduced blood pressure responses during passive heating, during both forms of heating the prolactin response was the same (active, 14.9 +/- 12.6 ng ml(-1); passive, 13.3 +/- 9.6 ng ml(-1); n.s.). These results suggest that thermoregulatory, i.e. core temperature, and not cardiovascular afferents provide the key stimulus for the release of prolactin, an indirect marker of central fatigue, during exercise in the heat.

Adult↗

Heat shock response of Neurospora crassa: protein synthesis and induced thermotolerance.

At elevated temperatures, germinating conidiospores of Neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 Mr and one minor heat shock protein of 30,000 Mr. Postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 Mr and a minor heat shock protein of 34,000 Mr. The three heat shock proteins of lower molecular weight are associated with mitochondria. This exclusive synthesis of heat shock proteins is transient, and after 60 min of exposure to high temperatures, restoration of the normal pattern of protein synthesis is initiated. Despite the transiency of the heat shock response, spores incubated continuously at 45 degrees C germinate very slowly and do not grow beyond the formation of a germ tube. The temperature optimum for heat shock protein synthesis is 45 degrees C, but spores incubated at other temperatures from 40 through 47 degrees C synthesize heat shock proteins at lower rates. Survival was high for germinating spores exposed to temperatures up to 47 degrees C, but viability declined markedly at higher temperatures. Germinating spores survived exposure to the lethal temperature of 50 degrees C when they had been preexposed to 45 degrees C; this thermal protection depends on the synthesis of heat shock proteins, since protection was abolished by cycloheximide. During the heat shock response mitochondria also discontinue normal protein synthesis; synthesis of the mitochondria-encoded subunits of cytochrome c oxidase was as depressed as that of the nucleus-encoded subunits.

Cell Survival↗

(CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene.

Previous analysis of the hsp26 gene of Drosophila melanogaster has shown that in addition to the TATA box and the proximal and distal heat shock elements (HSEs) (centered at -59 and -340, relative to the start site of transcription), a segment of (CT)n repeats at -135 to -85 is required for full heat shock inducibility (R.L. Glaser, G.H. Thomas, E.S. Siegfried, S.C.R. Elgin, and J.T. Lis, J. Mol. Biol. 211:751-761, 1990). This (CT)n element appears to contribute to formation of the wild-type chromatin structure of hsp26, an organized nucleosome array that leaves the HSEs in nucleosome-free, DNase I-hypersensitive (DH) sites (Q. Lu, L.L. Wallrath, B.D. Allan, R.L. Glaser, J.T. Lis, and S.C.R. Elgin, J. Mol. Biol. 225:985-998, 1992). Inspection of the sequences upstream of hsp26 has revealed an additional (CT)n element at -347 to -341, adjacent to the distal HSE. We have analyzed the contribution of this distal (CT)n element (-347 to -341), the proximal (CT)n element (-135 to -85), and the two HSEs both to the formation of the chromatin structure and to heat shock inducibility. hsp26 constructs containing site-directed mutations, deletions, substitutions, or rearrangements of these sequence elements have been fused in frame to the Escherichia coli lacZ gene and reintroduced into the D. melanogaster genome by P-element-mediated germ line transformation. Chromatin structure of the transgenes was analyzed (prior to gene activation) by DNase I or restriction enzyme treatment of isolated nuclei, and heat-inducible expression was monitored by measuring beta-galactosidase activity. The results indicate that mutations, deletions, or substitutions of either the distal or the proximal (CT)n element affect the chromatin structure and heat-inducible expression of the transgenes. These (CT)n repeats are associated with a nonhistone protein(s) in vivo and are bound by a purified Drosophila protein, the GAGA factor, in vitro. In contrast, the HSEs are required for heat-inducible expression but play only a minor role in establishing the chromatin structure of the transgenes. Previous analysis indicates that prior to heat shock, these HSEs appear to be free of protein. Our results suggest that GAGA factor, an abundant protein factor required for normal expression of many Drosophila genes, and heat shock factor, a specific transcription factor activated upon heat shock, play distinct roles in gene regulation: the GAGA factor establishes and/or maintains the DH sites prior to heat shock induction, while the activated heat shock factor recognizes and binds HSEs located within the DH sites to trigger transcription.

Animals↗

Expression of heat shock protein 70 is altered by age and diet at the level of transcription.

Because heat shock proteins have been shown to play a critical role in protecting cells from hyperthermia and other types of physiological stresses, it was of interest to determine what effect age and caloric restriction have on the ability of cells to regulate the expression of heat shock protein 70 (hsp70), the most prominent and most evolutionarily conserved of the heat shock proteins. Caloric restriction is the only experimental manipulation known to retard aging and increase survival of mammals. The ability of hepatocytes isolated from young/adult (4- to 7-month-old) and old (22- to 28-month-old) male Fischer F344 rats fed ad libitum or a caloric restriction diet (60% of the content of the ad libitum diet) to express hsp70 was determined after a mild heat shock (42.5 degrees C for 30 min). We found that the induction of hsp70 synthesis and mRNA levels by heat shock was 40 to 50% lower in hepatocytes isolated from old rats than in hepatocytes isolated from young rats. Using in situ hybridization, we found that essentially all hepatocytes from the young/adult and old rats expressed hsp70 in response to heat shock; therefore, the age-related decrease in the induction of hsp70 expression was not due to an age-related accumulation of cells that do not respond to heat shock. Measurements of hsp70 mRNA stability and hsp70 transcription demonstrated that the age-related decline in hsp70 expression arose from a decline in hsp70 transcription. Interestingly, the age-related decline in the induction of hsp70 expression was reversed by caloric restriction; e.g., the induction of hsp70 synthesis, mRNA levels, and nuclear transcription were significantly higher in hepatocytes isolated from old rats fed the caloric restricted diet than in hepatocytes isolated from old rats fed ad libitum. The levels of the heat shock transcription factor in nuclear extracts isolated from heat-shocked hepatocytes were measured in a gel shift assay. Binding of the heat shock transcription factor to the heat shock element decreased with age and was significantly higher in hepatocyte extracts isolated from old rats fed the caloric restriction diet than in those from old rats fed ad libitum. Thus, our study demonstrates that the ability of hepatocytes to respond to hyperthermia and express hsp70 decreases significantly with age and that this decrease occurs at the transcriptional level. In addition, caloric restriction, which retards aging, reversed the age-related decline in the induction of hsp70 transcription in hepatocytes.

Aging↗

Heat shock-induced translational control of HSP70 and globin synthesis in chicken reticulocytes.

Incubation of chicken reticulocytes at elevated temperatures (43 to 45 degrees C) resulted in a rapid change in the pattern of protein synthesis, characterized by the decreased synthesis of normal proteins, e.g., alpha and beta globin, and the preferential and increased synthesis of only one heat shock protein, HSP70. The repression of globin synthesis was not due to modifications of globin mRNA because the level of globin mRNA and its ability to be translated in vitro were unaffected. The HSP70 gene in reticulocytes was transcribed in non-heat-shocked cells, yet HSP70 was not efficiently translated until the cells had been heat shocked. In non-heat-shocked reticulocytes, HSP70 mRNA was a moderately abundant mRNA present at 1 to 2% of the level of globin mRNA. The rapid 20-fold increase in the synthesis of HSP70 after heat shock was not accompanied by a corresponding increase in the rate of transcription of the HSP70 gene or accumulation of HSP70 mRNA. These results suggest that the elevated synthesis of HSP70 is due to the preferential utilization of HSP70 mRNA in the heat-shocked reticulocyte. The heat shock-induced alterations in the reticulocyte protein-synthetic apparatus were not reversible. Upon return to control temperatures (37 degrees C), heat-shocked reticulocytes continued to synthesize HSP70 at elevated levels whereas globin synthesis continued to be repressed. Despite the presence of HSP70 mRNA in non-heat-shocked reticulocytes, we found that continued transcription was necessary for the preferential translation of HSP70 in heat-shocked cells. Preincubation of reticulocytes with the transcription inhibitor actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole blocked the heat shock-induced synthesis of HSP70. Because the level of HSP70 mRNA was only slightly diminished in cells treated with actinomycin D, we suggest two possible mechanisms for the preferential translation of HSP70 mRNA: the translation of only newly transcribed HSP70 mRNA or the requirement of a newly transcribed RNA-containing factor.

Animals↗