Ascorbic acid decreases heat shock protein 70 and plasma corticosterone response in broilers (Gallus gallus domesticus) subjected to cyclic heat stress

It is known that ascorbic acid (AA) supplementation can ameliorate the chicken's responses to heat stress. The influence of AA on heart heat shock protein 70 (hsp70) and plasma corticosterone (CS) was evaluated in young male broiler chickens fed either no AA (N-AA) or 500 mg AA /kg (AA) and exposed to cyclic high temperatures (21 to 30 to 21 degrees C) over a 3.5 h period on three consecutive days. Dietary AA supplementation elevated plasma AA and maintained it at high levels after heating, but in N-AA birds, only heat elevated plasma AA. In N-AA fed chickens, plasma CS was elevated and was further increased by heat stress as compared with AA-fed birds. Heart hsp70 expression was greater in N-AA-fed chickens compared to AA-fed chickens, and heat stress further elevated hsp70 in both N-AA- and AA-fed birds. The hsp70 increase after heat was two-fold greater in N-AA- vs. AA-fed birds. Plasma CS and heart hsp70 were positively correlated, plasma AA and heart hsp70 were negatively correlated, and plasma CS and AA were negatively correlated. It was concluded that chickens experience a less severe stress response after exposure to high temperatures when they are provided dietary AA.     

Influence of organic selenium on hsp70 response of heat-stressed and enteropathogenic Escherichia coli-challenged broiler chickens (Gallus gallus)

The effect of dietary selenium yeast, a source of organic selenium, on heat shock protein 70 (hsp70) responses, redox status, growth and feed utilization were evaluated either in enteropathogenic Escherichia coli-challenged (EPEC) or in heat-stressed (HS) male broiler chickens grown to 42 days of age. One day-old chicks in experiment 1 were challenged orally with EPEC (10(6) cfu/chicken on day 1 and boosted by water application on days 2, 3, and 4) and fed diets with or without selenium yeast. Body weight (BW), feed conversion ratio (FCR), and total mortality were determined at 42 days of age, and this was followed by collection of ileal tissue for the quantification of total glutathione (TGSH), reduced glutathione (GSH), oxidized glutathione (GSSG), and hsp70 in randomly selected chickens from each treatment. In experiment 2, male broiler chickens were fed diets with or without selenium yeast under a thermoneutral rearing condition. At four weeks of age, blood and hepatic tissue were collected from chickens maintained in the thermoneutral environment and from chickens subjected to HS (40 degrees C for 1 h) and analyzed for TGSH, GSH, GSSG, and hsp70. Selenium yeast improved BW, FCR, and decreased mortality in both control and EPEC-challenged chicks. Selenium yeast significantly attenuated hsp70 expression in EPEC-challenged chickens and in those subjected to HS. The EPEC challenge increased TGSH and GSSG levels and decreased GSH/GSSG ratio. However, GSSG level accumulated in chickens fed diets without selenium supplementation resulting in a lower GSH/GSSG ratio in the selenium yeast-fed group. Heat stress increased GSSG level and decreased GSH/GSSG ratio. Selenium yeast-fed groups maintained higher levels of GSSG before and after HS with a resultant lower GSH/GSSG ratio. The hsp70 response was significantly less in those chickens fed selenium yeast and challenged with either EPEC or HS than in those chickens given no supplemental selenium. The results of this study suggest that selenium yeast supplementation had imparted resistance to oxidative stress associated with enteric bacteria infection and to high temperature exposure. It is believed that the resistance to the stressors was due to an improved redox status of the selenium yeast-fed chickens.     

Expression of heat shock protein in broiler embryo tissues after acute cold or heat stress

This study evaluated the expression of heat shock protein 70 kD (hsp70) in broiler chicken embryos subjected to cold (Experiment I) or high incubation temperature (Experiment II). In each experiment, fertile eggs were distributed in three incubators kept at 37.8 degrees C. At day 13 (D13), D16, and D19 of incubation, the embryos were subjected to acute cold (32 degrees C) or heat (40 degrees C) for 4-6 hr. Immediately after cold or heat exposure, samples from the liver, heart, breast muscle, brain, and lungs of 40 embryos were taken per age and treatment (control or stressed embryos). A tissue pool from 10 embryos was used as 1 replication. The levels of hsp70 in each tissue sample was quantified by Western blot analysis. The data were analyzed in a 3 x 2 factorial arrangement of treatments with four replications. hsp70 was detected in all embryo tissues, and the brain contained 2- to 5-times more hsp70 protein compared to the other tissues in either cold or heat stressed embryos. hsp70 increases were observed in the heart and breast muscle of cold stressed embryos at D16 and D19, respectively. Heat stressed embryos showed an increase of hsp70 in the heart at D13 and D19, and in the lung at D19 of incubation. Younger embryos had higher hsp70 synthesis than older embryos, irrespective of the type of thermal stressor. The results indicate that the expression of hsp70 in broiler chicken embryos is affected by cold and heat distress, and is tissue- and age-dependent.     

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.     

Calmodulin is involved in heat shock signal transduction in wheat

The involvement of calcium and calcium-activated calmodulin (Ca(2+)-CaM) in heat shock (HS) signal transduction in wheat (Triticum aestivum) was investigated. Using Fluo-3/acetoxymethyl esters and laser scanning confocal microscopy, it was found that the increase of intracellular free calcium ion concentration started within 1 min after a 37 degrees C HS. The levels of CaM mRNA and protein increased during HS at 37 degrees C in the presence of Ca(2+). The expression of hsp26 and hsp70 genes was up-regulated by the addition of CaCl(2) and down-regulated by the calcium ion chelator EGTA, the calcium ion channel blockers LaCl(3) and verapamil, or the CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and chlorpromazine. Treatment with Ca(2+) also increased, and with EGTA, verapamil, chlorpromazine, or trifluoperazine decreased, synthesis of HS proteins. The temporal expression of the CaM1-2 gene and the hsp26 and hsp70 genes demonstrated that up-regulation of the CaM1-2 gene occurred at 10 min after HS at 37 degrees C, whereas that of hsp26 and hsp70 appeared at 20 min after HS. A 5-min HS induced expression of hsp26 after a period of recovery at 22 degrees C after HS at 37 degrees C. Taken together, these results indicate that Ca(2+)-CaM is directly involved in the HS signal transduction pathway. A working hypothesis about the relationship between upstream and downstream of HS signal transduction is presented.     

The effects of heat shock and acclimation temperature on hsp70 and hsp30 mRNA expression in rainbow trout: in vivo and in vitro comparisons

Heat shock (25° C) of 10° C-acclimated rainbow trout Oncorhynchus mykiss led to increases in heat shock protein 70 (hsp70) mRNA in blood, brain, heart, liver, red and white muscle, with levels in blood being amongst the highest. Hsp30 mRNA also increased with heat shock in all tissues with the exception of blood. When rainbow trout blood was heat shocked in vitro , both hsp70 and hsp30 mRNA increased significantly. In addition, these in vitro experiments demonstrated that blood from fish acclimated to 17° C water had a lower hsp70 mRNA heat shock induction temperature than did 5° C acclimated fish (20 v. 25° C). The hsp30 mRNA induction temperature (25° C), however, was unaffected by thermal acclimation. While increases in hsp70 mRNA levels in blood may serve as an early indicator of temperature stress in fish, tissue type, thermal history and the particular family of hsp must be considered when evaluating stress by these molecular means.     

Examination of heat shock protein mRNA accumulation in early Xenopus laevis embryos

Elevation of the incubation temperature of Xenopus laevis neurulae from 22 to 33-35 degrees C induced the accumulation of heat shock protein (hsp) 70 mRNA (2.7 kilobases (kb)) and a putative hsp 87 mRNA (3.2 kb). While constitutive levels of both hsp mRNAs were detectable in unfertilized eggs and cleavage-stage embryos, heat-induced accumulation was not observed until after the mid-blastula stage. Exposure of Xenopus laevis embryos to other stressors, such as sodium arsenite or ethanol, also induced a developmental stage-dependent accumulation of hsp 70 mRNA. To characterize the effect of temperature on hsp 70 mRNA induction, neurulae were exposed to a range of temperatures (27-37 degrees C) for 1 h. Heat-induced hsp 70 mRNA accumulation was first detectable at 27 degrees C, with relatively greater levels at 30-35 degrees C and lower levels at 37 degrees C. A more complex effect of temperature on hsp 70 mRNA accumulation was observed in a series of time course experiments. While continuous exposure of neurulae to heat shock (27-35 degrees C) induced a transient accumulation of hsp 70 mRNA, the temporal pattern of hsp 70 mRNA accumulation was temperature dependent. Exposure of embryos to 33-35 degrees C induced maximum relative levels of hsp 70 mRNA within 1-1.5 h, while at 30 and 27 degrees C peak hsp 70 mRNA accumulation occurred at 3 and 12 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of continuous heat stress on cell growth and protein synthesis in Aedes albopictus

Aedes albopictus (clone C6/36) cells, which normally grow at 28 degrees C, were maintained at a supraoptimal temperature of 37 degrees C. The effect of continuous heat stress (37 degrees C) on cell growth was analyzed as were the modifications occurring with protein synthesis during short- and long-term heat stress. We observed that cells in lag or exponential growth phase, present inhibition of cell growth, and cells in the lag phase showed more sensitivity to death than cells growing exponentially. During the first hour of exposing the cells to 37 degrees C, they synthesized two heat shock proteins (hsps) of 82 kd and 70 kd, respectively, concomitant with inhibition of normally produced proteins at control temperature (28 degrees C). However, for incubations longer than 2 hr at 37 degrees C, a shift to the normal pattern of protein synthesis occurred. During these transitions, two other hsps of 76 kd and 90 kd were synthesized. Pulse chase experiments showed that the 70-kd hsp is stable at least for 18 hr, when the cells are returned to 28 degrees C. However, if cells were incubated at 37 degrees C, the 70-kd hsp is stable for at least 48 hr. The 70-kd hsp was localized in the cytoplasmic and in the nuclear compartment. Our results indicate a possible role of hsp 70-kd protein in the regulation of cell proliferation.     

The gene expression response of the catadromous perciform barramundi Lates calcarifer to an acute heat stress

The acute heat-shock response of the tropical estuarine fish species barramundi Lates calcarifer as indicated by the expression of genes within stress (hsp 90AA, hsp 90AB, hsp 70 and hsc 70), metabolic (cisy, cco II and ldh) and growth (igf1 and mstn 1) related pathways was examined following an increase in water temperature from 28 to 36° C over 30 min. Lates calcarifer were maintained at the acute stress temperature of 36° C for 1 h before being returned to 28° C and allowed to recover at this temperature for a further 2 weeks. Muscle tissue sampling over the experimental period allowed for the expression quantification of stress, metabolic and growth-related genes via quantitative real-time polymerase chain reaction (qrt-PCR) where a robust and reliable normalization approach identified both α-tub and Rpl8 as appropriate genes for the analysis of gene expression in response to an acute heat stress. hsp90AA and hsp70 of the inducible heat-shock response pathway showed a massive up-regulation of gene expression in response to heat stress, whilst the constitutive heat-shock genes hsp90AB and hsp70 showed no change over the course of the experiment and a small increase after 2 weeks of recovery, respectively. Of the three genes representing the metabolic pathway (cisy, cco II and ldh) only cco II changed significantly showing a decrease in gene expression, which may suggest a small suppression of aerobic metabolism. igf1 of the growth pathway showed no significant differences in response to an acute heat stress, whilst mstn1 increased at the beginning of the heat stress but returned to basal levels soon after. Overall, the results demonstrate that an acute heat stress in L. calcarifer caused a significant increase in the expression of genes from the stress response pathway and a possible decrease in aerobic metabolism with only relatively minor changes to the growth pathway highlighting the hardy nature of L. calcarifer and its resilience in coping with sudden temperature changes routinely encountered within its natural environment.     

T lymphocyte stress response. II. Protection of translation and DNA replication against some forms of stress by prior hyperthermic stress

We have compared the effects of a mild heat shock and febrile temperatures on heat-shock protein (hsp) synthesis and development of stress tolerance in T lymphocytes. Our previous studies demonstrated that febrile temperatures (less than or equal to 41 degrees C) induced the synthesis of hsp110, hsp90, and the constitutive or cognate form of hsp70 (hscp70; a weak induction of the strongly stress-induced hsp70 was also observed. In the studies reported herein, we demonstrate that a mild heat shock (42.5 degrees C) reverses this ratio; that is, hsp70 and not hscp70 is the predominate member of this family synthesized at this temperature. Modest heat shock also enhanced the synthesis of hsp110 and hsp90. In order to assess the relationship between hsp synthesis and the acquisition of thermotolerance, purified T cells were first incubated at 42.5 degrees C (induction temperature) and then subsequently subjected to a severe heat-shock challenge (45 degrees C, 30 min). T cells first incubated at a mild heat-shock temperature were capable of total protein synthesis at a more rapid rate following a severe heat shock than control cells (induction temperature 37 degrees C). This phenomenon, which has been previously termed translational tolerance, did not develop in cells incubated at the febrile temperature (induction temperature 41 degrees C). Protection of translation also extended to immunologically relevant proteins such as interleukin-2 and the interleukin-2 receptor. Because clonal expansion is a critical event during an immune response, the effects of hyperthermic stress on DNA replication (mitogen-induced T cell proliferation) was also evaluated in thermotolerant T cells. DNA synthesis in control cells (induction temperature 37 degrees C) was severely inhibited following heat-shock challenge at 44 degrees C or 45 degrees C; in contrast, T cells preincubated at 42.5 degrees C rapidly recovered their DNA synthetic capacity. T cells preincubated at a febrile temperature were moderately protected against hyperthermic stress. The acquisition of thermotolerance was also associated with enhanced resistance to chemical (ethanol)-induced stress but not to heavy metal toxicity (cadmium) or dexamethasone-induced immunosuppression. These studies suggest that prior hsp synthesis may protect immune function against some forms of stress (e.g., febrile episode) but would be ineffective against others such as elevated glucocorticoid levels which normally occur during an immune response.     

The 70-kDa heat shock protein response in two intertidal sculpins,Oligocottus maculosus and O. snyderi: relationship of hsp70 and thermal tolerance

The role that hsp70 plays in influencing thermal tolerance of a whole animal is not clearly understood. We explored this question by examining liver hsp70 response in the tidepool sculpin (Oligocottus maculosus) and fluffy sculpin (O. snyderi), which have distinct distribution patterns in the intertidal zone. The tidepool sculpin is in upper and lower tidepools, while the fluffy sculpin is exclusively in lower tidepools during a low tide. We conducted experiments in order to investigate: (1) habitat water temperatures; (2) upper thermal tolerance limits; (3) the cellular hsp70 response to changes in water temperature in nature; (4) induction temperatures for hepatic hsp70 and hsp70 mRNA; and (5) effects of long-term heat stress on liver hsp70 levels, in these sculpins. Accordingly, we found: (1) the tidepool sculpin was exposed to a wider temperature range in nature; (2) the tidepool sculpin had higher lethal and induction temperatures for hsp70; (3) the liver hsp70 level of the tidepool sculpin was less sensitive to changes in water temperatures; and (4) the tidepool sculpin had higher constitutive hsp70 levels in nature, compared with the fluffy sculpin. From these results, we proposed that the less thermally sensitive tidepool sculpin may enhance its thermal tolerance by having a large pool of cellular hsp70, thus allowing it to inhabit the upper intertidal zone with relatively large and unpredictable fluctuations in environmental variables.     

Acute heat stress induces oxidative stress in broiler chickens

The stress responses and possible oxidative damage in plasma, liver and heart were investigated in broiler chickens acutely exposed to high temperature. Eighty 5-week old broiler chickens were exposed to 32 degrees C for 6h. The extent of lipid peroxidation, activities of superoxide dismutase and total antioxidant power in plasma, liver and heart tissues were investigated. Meanwhile, the blood metabolites such as glucose, urate, triiodothyronine, thyroxine, corticosterone, ceruloplasmin and creatine kinase were measured before and after 3 and 6h of heat exposure. The results showed that oxidative stress could be induced in 5-week old broiler chickens by acute heat exposure (32 degrees C, 6h). The results suggest that the elevated body temperature can induce the metabolic changes that are involved in the induction of oxidative stress. The liver is more susceptible to oxidative stress than heart during acute heat exposure in broiler chickens. The oxidative stress should be considered as part of the stress response of broiler chickens to heat exposure.     

Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure

The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (+15 degrees C for 1 h) or exposed to either CuSO(4) (200 microM for 24 h), CdCl(2) (10 microM for 24 h) or NaAsO(2) (50 microM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish.