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.     

Antioxidant responses of Chilo suppressalis (Lepidoptera: Pyralidae) larvae exposed to thermal stress

High temperatures cause a variety of physiological stress responses in insects, including increased generation of reactive oxygen species (ROS), which can cause oxidative damage. This study investigated the effects of thermal stress on ROS generation, the expression of heat shock protein 70 (Hsp70) at the mRNA and protein levels, the activity of antioxidant enzymes (SOD, CAT), and apoptosis in hemocytes of Chilo suppressalis larvae. Results indicated that thermal stress significantly elevated the level of ROS and antioxidant enzyme activity in C. suppressalis larvae. Real-time quantitative PCR showed that hsp70 gene expression was induced by heat stress. Flow cytometric results revealed that the expression profile of Hsp70 at the protein level was in agreement with that at the mRNA level. The expression of Hsp70 at both the mRNA and protein levels reached a maximum at 36 °C in larval hemocytes. Exposure to tested temperatures did not cause any significant change in the rate of apoptosis in larval hemocytes. These results suggest that thermal stress leads to oxidative stress and that antioxidant enzymes and the Hsp70 play an important role in reducing oxidative damage in C. suppressalis larvae.     

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.     

Examination of the expression of the heat shock protein gene, hsp110, in Xenopus laevis cultured cells and embryos

Eukaryotic organisms respond to various stresses with the synthesis of heat shock proteins (HSPs). HSP110 is a large molecular mass HSP that is part of the HSP70/DnaK superfamily. In this study, we have examined, for the first time, the expression of the hsp110 gene in Xenopus laevis cultured cells and embryos. Sequence analysis revealed that the protein encoded by the hsp110 cDNA exhibited 74% identity with its counterparts in mammals and only 27-29% with members of the Xenopus HSP70 family. Hsp110 mRNA and/or protein was detected constitutively in A6 kidney epithelial cells and was inducible by heat shock, sodium arsenite, and cadmium chloride. However, treatment with ethanol or copper sulfate had no detectable effect on hsp110 mRNA levels. Similar results were obtained for hsp70 mRNA except that it was inducible with ethanol. In Xenopus embryos, hsp110 mRNA was present constitutively during development. Heat shock-inducible accumulation of hsp110 mRNA occurred only after the midblastula stage. Whole mount in situ hybridization analysis revealed that hsp110 mRNA accumulation in control and heat shocked embryos was enriched in selected tissues. These studies demonstrate that Xenopus hsp110 gene expression is constitutive and stress inducible in cultured cells and developmentally- and tissue specifically-regulated during early embryogenesis.     

Hsp 70 expression and function during gametogenesis

The dramatic transformations in nuclear content and cellular organization that occur during gametogenesis require unique regulation and execution of the mitotic and meiotic cell cycle, apoptotic cell death, DNA recombination and repair, and cellular differentiation. These processes are accompained by the constitutive and developmentally regulated expression of a number of hsp70 genes encoding 70 kDa heat shock proteins (Hsp70), including several hsp70s whose expression is unique to male germ cells. Examining the expression and function of Hsp70s in germ cells has provided significant insights into mechanisms of hsp70 gene regulation and Hsp70 protein function, as well as the developmental processes of gametogenesis.     

Geographic variation in thermal tolerance and strategies of heat shock protein expression in the land snail <Emphasis Type="Italic">Theba pisana</Emphasis> in relation to genetic structure

Land snails are exposed to conditions of high ambient temperature and low humidity, and their survival depends on a suite of morphological, behavioral, physiological, and molecular adaptations to the specific microhabitat. We tested in six populations of the land snail Theba pisana whether adaptations to different habitats affect their ability to cope with thermal stress and their strategies of heat shock protein (HSP) expression. Levels of Hsp70 and Hsp90 in the foot tissue were measured in field-collected snails and after acclimation to laboratory conditions. Snails were also exposed to various temperatures (32 up to 54 °C) for 2 h and HSP messenger RNA (mRNA) levels were measured in the foot tissue and survival was determined. To test whether the physiological and molecular data are related to genetic parameters, we analyzed T. pisana populations using partial sequences of nuclear and mitochondrial DNA ribosomal RNA genes. We show that populations collected from warmer habitats were more thermotolerant and had higher constitutive levels of Hsp70 isoforms in the foot tissue. Quantitative real-time polymerase chain reaction (PCR) analysis indicated that hsp70 and hsp90 mRNA levels increased significantly in response to thermal stress, although the increase in hsp70 mRNA was larger compared to hsp90 and its induction continued up to higher temperatures. Generally, warm-adapted populations had higher temperatures of maximal induction of hsp70 mRNA synthesis and higher upper thermal limits to HSP mRNA synthesis. Our study suggests that Hsp70 in the foot tissue of T. pisana snails may have important roles in determining stress resistance, while Hsp90 is more likely implicated in signal transduction processes that are activated by stress. In the phylogenetic analysis, T. pisana haplotypes were principally divided into two major clades largely corresponding to the physiological ability to withstand stress, thus pointing to genetically fixed tolerance.     

Physiological consequences of the supralittoral fringe: microhabitat temperature profiles and stress protein levels in the tropical periwinkle Cenchritis muricatus (Linneaus, 1758)

Habitat selection by marine snails is profoundly affected by variations in biotic and abiotic factors. In the supralittoral fringe of Caribbean rocky shores, the littorinid Cenchritis muricatus endures a near-terrestrial existence through a combination of active microhabitat choice and, during dry periods, repose. In this study, we sought to compare knobby periwinkle body size, thermal load, water loss, and stress protein expression among common supralittoral microhabitats to determine the physiological consequences of habitat selection. In this study, we show: (1) body temperatures in these snails exhibit daily fluctuations of more than 20°C and regularly exceed 46°C, (2) microhabitats differ in thermal stress over small spatial scales, with snails on black rocks and within crevices experiencing more extreme temperatures than snails on white rocks or grass, (3) water losses of 8.5% of total snail mass do not affect survival during 1 week, and (4) Hsp70, but not Hsp90, expression varies slightly among microhabitats but at a level much lower than physiologically possible. During arousal following hydration, snails exhibited substantially higher levels of Hsp70s than snails on dry substrates in the field. When inactive, Cenchritis appears to utilize a distinctly different physiological state consistent with aestivation metabolism and does not exhibit significant up-regulation of inducible heat shock proteins (Hsps). In summary, studies lacking detailed thermal and hydration history, and relying only upon Hsp levels, may misrepresent the true physiological consequences of microhabitat choice for high-shore tropical gastropods.     

Analysis of the variation in the hsp70-1 and hsp90alpha mRNA expression in human myocardial tissue that has undergone surgical stress

In the present work we reported a semiquantitative detection of messenger ribonucleic acids (mRNAs) encoding the human heat shock proteins Hsp70-1, the stress inducible member of the HSP70 family, and hsp90alpha, the inducible member of the HSP90 family. We investigated the change in the expression of these mRNAs in tissue samples taken from the right atrium of 48 pediatric patients, soon after the ischemic period during surgery to correct congenital heart diseases, in which a crystalloid cold cardioplegic solution was used. No significant variations were found for either hsp70-1 or hsp90alpha expressions. Moreover, we searched for an association between the hsp70-1 promoter region polymorphism and the expression of the hsp70-1 in a smaller group of these patients (n = 27). The -110AA genotype was on average significantly associated with a decrease in the hsp70-1 mRNA level (P < 0.05), whereas the other genotypes -110AC or -110CC did not seem to be associated with the hsp70-1 expression level. The lack of any observed increase in the hsp70-1 expression level may be due to the high basal level of the Hsp70 protein in the tissues examined.