Heat shock protein induction in the freshwater prawn Macrobrachium malcolmsonii: acclimation-influenced variations in the induction temperatures for Hsp70

The intracellular build-up of thermally damaged proteins following exposure to heat stress results in the synthesis of heat shock proteins (Hsps). In the present study, the upper thermal tolerance and expression of heat shock protein 70 (Hsp70) were examined in juveniles of the freshwater prawn Macrobrachium malcolmsonii that had been acclimated at two different temperatures, i.e. 20 degrees C (group A) and 30 degrees C (group B), in the laboratory for 30 days. Upper thermal tolerance was determined by a standard method. For heat-shock experiments, prawns in groups A and B were exposed to various elevated temperatures for 3 h each, followed by 1 h recovery at the acclimation temperature. Endogenous levels of Hsp70 were determined in the gill, heart, hepatopancreas and skeletal muscle tissues by Western blotting analysis of one dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The critical thermal maximum (CT max) for prawns in groups A and B was 37.7+/-0.27 degrees C and 41.41+/-0.16 degrees C, respectively. In general, Western blotting analysis for Hsp70 revealed one band at the 70 kDa region, containing both constitutive (Hsc70) and inducible (Hsp70) isoforms, in the gill and heart tissues; these were not detected in the hepatopancreas and skeletal muscle tissues. The onset temperature for Hsp70 induction in both gill and heart tissues was 30 degrees C for prawns in group A and 34 degrees C for those in group B. The optimum induction temperatures (at which Hsp70 induction was maximum) were found to be 34 degrees C and 32 degrees C, respectively, in the gill and heart tissues of group A prawns, and 38 degrees C and 36 degrees C, respectively, for group B prawns. These results suggest that the temperature at which acclimation occurs influences both upper thermal tolerance and Hsp70 induction in M. malcolmsonii.     

Effects of the natural tidal cycle and artificial temperature cycling on Hsp levels in the tidepool sculpin Oligocottus maculosus

The rocky intertidal zone is characterized by a predictable cycle of environmental change cued by the ebb and flow of the tides. Tidepools are thus an excellent environment in which to determine whether predictability of environmental change can entrain an endogenous rhythmicity in heat shock protein (Hsp) levels. In this study, we monitored changes in Hsp mRNA and protein levels that occurred over the tidal cycle in tidepool sculpins and investigated whether there was an endogenous tidal rhythm in Hsp expression that persisted once the sculpins were transferred to a stable environment. Fluctuations in the tidepool environment increased hsc70, hsp70, and hsp90 mRNA levels, which translated into increased Hsc/Hsp70 and Hsp90 protein levels; however, this was not due to an endogenous tidal rhythm in Hsp levels because sculpins held under constant conditions did not show any rhythmicity in the expression of these genes. By exposing sculpins to an artificial temperature cycling regime that mimicked the temperature changes of a mid-intertidal pool, we were able to account for the direct role of temperature in regulating Hsp expression. However, there are additional extrinsic factors that likely integrate with temperature and result in differences between the hsp induction profiles that were observed in sculpins inhabiting their natural environment and those in cycling conditions in the laboratory.     

Tissue-specific variations in the induction of Hsp70 and Hsp64 by heat shock in insects

The patterns of heat-induced synthesis (37 degrees C to 45 degrees C) of heat shock proteins (Hsps) in different tissues of grasshoppers and cockroaches from natural populations and in laboratory-reared gram-pest (Heliothis armigera) were examined by 35S-methionine labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography. Whereas 45 degrees C was lethal in most cases, optimal induction of Hsp synthesis was seen between 37 degrees C and 42 degrees C. The ongoing protein synthesis was not much affected at these temperatures, except in the tissues of adult H. armigera exposed to 42 degrees C. The profiles of the Hsps induced in the tissues of the insects, however, were different. From the relative abundance of the synthesis of 70-kDa (Hsp70) and 64-kDa (Hsp64) polypeptides, three categories of heat shock response were identified: (1) induction of abundant Hsp70 but little Hsp64 (malpighian tubules, male accessory glands, and ovaries of adult grasshoppers), (2) abundant Hsp64 but little Hsp70 (testes of adult grasshoppers, testes and malpighian tubules of adult cockroaches, and testes, malpighian tubules, and fat bodies of H. armigera larvae), and (3) induction of both Hsp70 and Hsp64 in more or less equal abundance (ovaries of adult cockroaches, salivary glands of H. armigera larvae, and malpighian tubules, male accessory glands, testes, and ovaries of adult H. armigera). Cockroaches collected from storerooms showed detectable synthesis of Hsp64 and/or Hsp70 only after heat shock, but those collected from drains showed detectable synthesis of both Hsp70 and Hsp64 in different tissues without heat stress. Western blotting showed that the 64-kDa polypeptide in these insects is a member of the Hsp60 family. Grasshopper testes, which synthesized negligible Hsp70 but abundant Hsp64 after heat shock, developed thermotolerance. Thus, heat shock response is modulated by developmental and environmental factors in different tissues of insects.     

Thermal acclimation and stress in the American lobster, Homarus americanus: equivalent temperature shifts elicit unique gene expression patterns for molecular chaperones and polyubiquitin

Using homologous molecular probes, we examined the influence of equivalent temperature shifts on the in vivo expression of genes coding for a constitutive heat shock protein (Hsc70), heat shock proteins (Hsps) (Hsp70 and Hsp90), and polyubiquitin, after acclimation in the American lobster, Homarus americanus. We acclimated sibling, intermolt, juvenile male lobsters to thermal regimes experienced during overwintering conditions (0.4 +/- 0.3 degrees C), and to ambient Pacific Ocean temperatures (13.6 +/- 1.2 degrees C), for 4-5 weeks. Both groups were subjected to an acute thermal stress of 13.0 degrees C, a temperature shift previously found to elicit a robust heat shock response in ambient-acclimated lobsters. Animals were examined after several durations of acute heat shock (0.25-2 hours) and after several recovery periods (2-48 hours) at the previous acclimation temperature, following a 2-hour heat shock. Significant inductions in Hsp70, Hsp90, and polyubiquitin messenger RNA (mRNA) levels were found for the ambient-acclimated group. Alternatively, for the cold-acclimated group, an acute thermal stress over an equivalent interval resulted in no induction in mRNA levels for any of the genes examined. For the ambient-acclimated group, measurements of polyubiquitin mRNA levels showed that hepatopancreas, a digestive tissue, incurred greater irreversible protein damage relative to the abdominal muscle, a tissue possessing superior stability over the thermal intervals tested.     

Evaluation of heat shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna minor

Heat shock proteins (Hsps) are known to be induced in response to short-term stress. The present study aimed to evaluate the potential of Hsp70 as a biomarker of stress produced by increased temperature, osmotic pressure, and exposure to cadmium and sodium chloride in marine macroalgae and fresh water plant species. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed with a working range of 0.025-10 μg ml^-1 using a monoclonal antibody raised against purified Hsp70 of Phaseolus aureus (mung bean). Fucus serratus (toothed wrack), Chondrus crispus (Stackhouse or Carrageen moss), Ulva lactuca (sea lettuce) and Lemna minor (common duckweed) sample extracts were stressed for up to 24 h and then tested in the IC-ELISA. The presence of Hsp70 and cross-reactivity of the monoclonal antibody was confirmed by Western blot. The heat shock response was confirmed in each species using a 2-h 42°C treatment. Following heat shock, Hsp70 concentrations increased to a peak at 2 h (F. serratus) or 4 h (L. minor), after which concentrations decreased. Osmotic and cadmium stresses also resulted in elevated Hsp70 concentrations in samples of F. serratus and L. minor when compared with unstressed controls. In both, osmotic and metal stress, the production of Hsp70 increased to a maximum and subsequently decreased as the stressor levels increased. Results suggest that Hsp70 IC-ELISA could potentially be applied to the detection of stress in these aquatic species, although it would probably be most effective when used in conjunction with other measurements to provide a stressor-specific biomarker profile or fingerprint.     

Lack of glucose and hsp70 responses in haddock Melanogrammus aeglefinus (L.) subjected to handling and heat shock

Juvenile haddock Melanogrammus aeglefinus ( c. 39 g) were exposed to either a handling stressor (1 min out of water) or heat shock (increase from 10 to 15° C for 1 h), and plasma cortisol, plasma glucose and gill hsp70 levels were determined before, and at 1, 3, 6, 12, 24 and 48 h post-stress. The pattern of cortisol increase was similar following both stressors, with levels increasing by 25-fold at 1 h post-stress, but returning to pre-stress levels (2–5 ng ml⁻¹) by 3 h. In contrast, neither handling nor heat shock caused an increase in plasma glucose levels. Although gill hsp70 was detected, presumably constitutive levels, in both control and heat shocked groups, there were not significant changes in gill hsp70 levels after exposure to heat shock. The lack of glucose and hsp70 responses to these typical stressors is consistent with previous studies on Atlantic cod Gadus morhua , and suggests that the stress physiology of Gadidae differs from the 'typical' teleost.     

Genetic heterogeneity of heat shock protein synthesis as a factor determining the resistance to stressors in mammalia

The relationship between the levels of 70 kDa family heat shock protein (Hsp) synthesis and lymphocyte sensitivity to stressors was investigated. Lymphocyte cultivation in mitogen deprived culture medium and/or the cell treatment with alkylating agents have been used as a stress challenge. Model experiments with two inbred murine strains genetically contrasting by the sensitivity to alkylating agents demonstrated that the basic level of Hsp synthesis depends on genotype. The quantity Hsp70 mRNA, as well as intracellular level of the proteins, in BALB/c was significantly higher than those in C57BL/6 mice. The mice, which were characterized by higher Hsp levels, demonstrated higher resistance to alkylating agent action. The induction of surplus amount of Hsp by heat shock increased the cell resistance to an alkylating agent melphalan. Lymphocyte isolated from high Hsp producers BALB/c mice were more resistant to apoptotic signals induced by mitogen deprivation.     

Evaluation of heat shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna minor

Heat shock proteins (Hsps) are known to be induced in response to short-term stress. The present study aimed to evaluate the potential of Hsp70 as a biomarker of stress produced by increased temperature, osmotic pressure, and exposure to cadmium and sodium chloride in marine macroalgae and fresh water plant species. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed with a working range of 0.025–10 μg?ml^?1 using a monoclonal antibody raised against purified Hsp70 of Phaseolus aureus (mung bean). Fucus serratus (toothed wrack), Chondrus crispus (Stackhouse or Carrageen moss), Ulva lactuca (sea lettuce) and Lemna minor (common duckweed) sample extracts were stressed for up to 24 h and then tested in the IC-ELISA. The presence of Hsp70 and cross-reactivity of the monoclonal antibody was confirmed by Western blot. The heat shock response was confirmed in each species using a 2-h 42°C treatment. Following heat shock, Hsp70 concentrations increased to a peak at 2 h (F. serratus) or 4 h (L. minor), after which concentrations decreased. Osmotic and cadmium stresses also resulted in elevated Hsp70 concentrations in samples of F. serratus and L. minor when compared with unstressed controls. In both, osmotic and metal stress, the production of Hsp70 increased to a maximum and subsequently decreased as the stressor levels increased. Results suggest that Hsp70 IC-ELISA could potentially be applied to the detection of stress in these aquatic species, although it would probably be most effective when used in conjunction with other measurements to provide a stressor-specific biomarker profile or fingerprint.     

Differential temperature dependency of chemical stressors in HSF1-mediated stress response in mammalian cells

Expression of stress proteins is generally induced by a variety of stressors. To gain a better understanding of the sensing and induction mechanisms of stress responses, we studied the effects of culture temperature on responses to various stressors, since the induction of hsp70 in mammalian cells by heat shock is somehow modulated by culture temperature. Hsp70 was not induced by treatment with sodium arsenite, azetidine-2-carboxylic acid, or zinc sulfate at the level of heat shock factor (HSF) 1 activation in cells incubated at low temperature, although these treatments induced hsp70 in cells incubated at 37 degrees C. The repression of sodium arsenite or zinc sulfate-induced HSF1 activation by low temperature was not simply due to the inhibition of protein synthesis. On the other hand, heat shock and iodoacetamide induced HSF 1 activation in cells incubated at either temperature. Thus, there seem to be two kinds of stressors that induce HSF1 activation independently of or dependent on culture temperature. Furthermore, the reduction of glutathione level seemed to be essential for HSF1 activation by chemical stressors.     

Effects of antineoplastic agents on cytoplasmic and membrane-bound heat shock protein 70 (Hsp70) levels

Here we report on the study of the effects of different antineoplastic agents, including cytarabine, 4-hydroperoxyifosfamide, the activated form of ifosfamide, vincristine, and paclitaxel, with regard to their capacity to modulate the amount of cytoplasmic and membrane-bound heat shock protein 70 (Hsp70). Hsp70 levels were measured in the myelogenous leukemic cell line K562, in the human colon carcinoma cell line CX2, and in peripheral blood lymphocytes (PBL) under physiological conditions (37 degrees C), and following non-lethal heat shock at 41.8 degrees C. A concentration of 1 microM and an incubation period of 2 h were determined as non-lethal, since none of the different antineoplastic agents induced necrosis or apoptosis in untreated or heat-shocked cells under these conditions. Our results show that tubulin-interacting agents, including vincristine and paclitaxel, but not DNA-interacting agents, including cytarabine and ifosfamide, selectively increase the amount of cytoplasmic Hsp70 in tumor and normal cells, as measured by semi-quantitative Western blot analysis. Mechanistically, a vincristine- and paclitaxel-induced tubulin assembly, as demonstrated by immunofluorescence microscopy, might be responsible for the elevated cytoplasmic Hsp70 levels. Interestingly, an increased membrane expression of Hsp70 following treatment with vincristine or paclitaxel was selectively observed on tumor cells, but not on normal cells.     

The effect of environmental salinity on the level of heat shock proteins in gill epithelium of Mytilus edilis L. mussel

The composition and the level of heat shock proteins in the gill epithelium cells of mussels Mytilus edulis L. from the White Sea under different levels of environmental salinity were studied by the method of immunoblotting. In mussels maintained under normal salinity (26%), constitutive Hsp70 and protein of about 40 kDa were revealed. After long-term (11?C14 days) acclimation to 14 and 35?? of the level Hsp70 in gill epithelium cells increased. Hsp70 induction was also observed in cells of isolated gills after salinity shock at 14% for 3 and 24 h.     

Genetic heterogeneity of heat shock protein synthesis as a factor determining the resistance of mammalia to stressors

The relationship between the levels of 70 kDa family heat shock protein (Hsp) synthesis and lymphocyte sensitivity to stressors was investigated. Lymphocyte cultivation in mitogen deprived culture medium and (or) the cell treatment with alkylating agents have been used as a stress challenge. On the model of two inbred murine strains genetically contrasting by the sensitivity to alkylating agents we succeeded in demonstration that the basic level of Hsp synthesis depends on genotype. The quantity Hsp mRNA, as well as the intracellular level of the proteins were significantly higher in BALB/c than in C57BL/6 mice. The mice characterized by higher Hsp levels demonstrated higher resistance to alkylating agent action. The induction of surplus amount of Hsp by heat shock increased the cell resistance to the alkylating agent melphalan. Lymphocyte isolated from high Hsp producers, BALB/c mice, were more resistant to apoptotic signals induced by mitogen deprivation.     

Heat shock protein (Hsp70) induced by a mild heat shock slightly moderates plasma osmolarity increases upon salinity transfer in rainbow trout (Oncorhynchus mykiss)

We have investigated whether mild heat shock, and resulting Hsp70 expression, can confer cross-protection against the stress associated with transfer from freshwater (FW) to seawater (SW) in juvenile rainbow trout (Oncorhynchus mykiss). In experimental Series I, juvenile trout reared in FW were transferred from 13.5 degrees C to 25.5 degrees C in FW, held for 2 h, returned to 13.5 degrees C for 12 h, and then transferred to 32 ppt SW at 13.5 degrees C. Branchial Hsp70 increased approximately 10-fold in the heat-shocked fish relative to the control by the end of recovery and remained high 2, 8, and 24 h post-salinity transfer. However, no clear differences could be detected in blood parameters (blood hemoglobin, hematocrit, MCHC, plasma Na(+) and plasma osmolarity) or muscle water content between heat-shocked and sham-shocked fish in SW at any sampling interval (0, 2, 8, 24, 48, 120, 240 and 360 h post-SW transfer). In experimental Series II, trout acclimated to 8 degrees C were heat-shocked at 22 degrees C for 2 h, allowed to recover 18 h, and exposed to a more severe salinity transfer (either 36 or 45 ppt) than in Series I. Branchial Hsp70 levels increased approximately 6-fold in heat-shocked fish, but had declined to baseline after 120 h in SW. Plasma osmolarity and chloride increased in both groups upon transfer to 36 ppt; however, the increase was significantly less in heat-shocked fish when compared to the increase observed in sham-shocked fish at 24 h. No significant differences could be detected in branchial Na(+)/K(+)-ATPase activity or Na(+)/K(+)-ATPase alpha1a and alpha1b mRNA expression between the two groups. Our data indicate that a mild temperature shock has only modest effects on the ability of rainbow trout to resist osmotic stress during FW to SW transfer.     

Insulin-induced myocardial protection in isolated ischemic rat hearts requires p38 MAPK phosphorylation of Hsp27

Six hours after insulin treatment, hearts express heat shock protein 70 (Hsp70) and have improved contractile function after ischemia-reperfusion injury. In this study we examined hearts 1 h after insulin treatment for contractile function and for expression of Hsp70 and Hsp27. Adult, male Sprague-Dawley rats were assigned to groups: 1) sham, 2) control, 3) insulin injected (200 microU/g body wt), 4) heat shock treated (core body temperature, 42 degrees C for 15 min), and 5) heat shock and insulin treated. At 1 h after these treatments, hearts were isolated, equilibrated to Langendorff perfusion for 30 min, and then subjected for 30 min no-flow global ischemia (37 degrees C) followed by 2 h of reperfusion. Insulin-treated hearts had significantly increased contractile function compared with control hearts. At 1 h after insulin treatment, a minimal change in Hsp70 and Hsp27 content were detected. By 3 h after insulin treatment, a significant increase in Hsp70, but not Hsp27, was detected by Western blot analysis. By immunofluorescence, minimal Hsp70 was detected in insulin-treated hearts, whereas Hsp27 was detected in all hearts, indicative of its constitutive expression. Phosphospecific isoforms of Hsp27 were detected in insulin-, heat shock-, and heat shock and insulin-treated hearts. After ischemia and reperfusion, the insulin-treated hearts had significantly elevated levels of phosphorylated Hsp27. Inhibition of p38 MAPK with SB-203580 blocked the insulin-induced phosphorylation of Hsp27 and the improved functional recovery. In conclusion, insulin induces an apparent rapid phosphorylation of Hsp27 that is associated with improved functional recovery after ischemia-reperfusion injury.     

Field and laboratory investigations of the thermal influence on tissue-specific Hsp70 levels in common carp (Cyprinus carpio).

Thermal discharge from power stations can affect normal environmental conditions and change in heat shock proteins expression of native fish with increasing temperature. In this study, we investigated levels of Hsp70 in the heart, kidney, brain and gill of the common carp Cyprinus carpio both in long-term heat discharge environment and after 24 h acute heat shock exposure. In laboratory exposure experiments, fish acclimated at 10 degrees C were exposed to various elevated temperatures (20, 24 and 28 degrees C). Hsp70 concentrations were determined in tissues by Western blotting analysis after one dimensional SDS-PAGE separation. In the field study, the level of Hsp70 in the gill of the carp remained at control values, and Hsp70 expression in the heart, kidney and brain underwent a 2.8 to 3.7-fold increase. A lower thermal sensitivity of the Hsp70 response of the brain, compared with the heart, kidney and gill, was observed in the laboratory experiments. Our data show that these tissues had different levels of Hsp70 responses to thermal influence both in acute exposure and long-term acclimation. The pattern of tissue Hsp70 expression may have a close relationship with the thermal tolerance of the carp and allows the fish to survive long-term thermal pollution.