Small heat shock proteins and adaptation of various Drosophila species to hyperthermia

The dynamics and the level of accumulation of small heat shock proteins (sHSP group 21–27) after a heat exposure were studied in three Drosophila species differing in thermotolerance. The southern species Drosophila virilis, having the highest thermotolerance, surpassed thermosensitive D. lummei and D. melanogaster in the level of sHSPs throughout the temperature range tested. The results suggest an important role of sHSPs in the molecular mechanisms of adaptation to adverse environmental conditions, particularly to hyperthermia.     

Patterns of protein synthesis following heat shock in pupae of Drosophila melanogaster

Pupae of Drosophila melanogaster were heat-shocked under conditions required to induce phenocopies in more than 90% of the flies that subsequently emerge. The effects of these treatments on protein synthesis in two tissues (thoracic epithelium and brain) were followed for several hours after the heat treatments. Results from pulse-labeling and protein separations on sodium dodecylsulfate (SDS) acrylamide gels showed a virtually complete cessation of protein synthesis immediately after the shock, followed by a noncoordinate resumption of the starting pattern. Similar experiments following double heat shocks demonstrated a more rapid resumption of synthesis of heat shock proteins after two successive heat treatments than after a single one.     

Effect of heat stress on development in vitro and in vivo and on synthesis of heat shock proteins in porcine embryos

The present study was conducted (1) to examine the effect of an acute increase in ambient temperature on the development of porcine day 6 embryos in culture and after transfer to recipient gilts, and (2) to analyze intracellular production of heat shock proteins (hsps). The viability of porcine day 6 embryos following a temporary acute elevation in ambient temperature (at 42°–45.5°C and for 10–180 min) was examined. Synthesis of 70 kDa hsp (hsp 70) and 90 kDa hsp (hsp90) was determined by SDS-PAGE and Western blot analysis in porcine day 6 embryos subjected to heat stresses. Nonheat-stressed embryos were considered as control. Significantly higher numbers of viable nuclei were observed in treatment groups of 42°C-10 min (236.6 ± 71.4; P < 0.05) and 43°C-30 min (276.8 ± 89.4; P < 0.005) compared to control (173.9 ± 53.9). The 42°C-180 min group (158.0 ± 27.1 μm) had a greater increase in diameter after 24 hr in culture following heat stress compared to control (82.5 ± 47.3 μm), while heat stress with 43°C for ≧60 min, 44°–44.5°C for ≧30 min, or 45°-45.5°C for ≧10 min impaired their survival, as assessed by differences in number of viable nuclei. The embryos subjected to heat stresses under the conditions of 42°C-180 min, 43°C-10 min, 43°C-30 min, 44°C-10 min, or 45°C-10 min developed to normal piglets after transfer to recipient gilts. Overall pregnancy rate was 75% (6/8), and farrowing rate 62.5% (5/8). Of heat-stressed embryos transferred, 59% (36/61) developed to normal piglets. Heat-stress conditions of 42°C for 180 min, 43°C for 30 min, 44°C for 10 min, and 45°C for 10 min were determined as critical with respect to the in vitro and in vivo survival of porcine embryos. Porcine day 6 embryos constitutively synthesized hsp70 even without heat stress, while hsp90 was detected only at trace level. Neither hsp70 nor hsp90 levels increased in the embryos subjected to heat stresses. In conclusion, porcine day 6 embryos could continue to develop in vivo or during in vitro culture after exposure to acute and temporary rise in temperature. However, no increase of hsp70 and hsp90 was observed in the heat-stressed porcine embryos, while hsp70 was detected in the nonheat-stressed porcine embryos. The precise mechanism of the thermotolerance was unclear. © 1996 Wiley-Liss, Inc.     

Changes in heat shock protein synthesis and heat sensitivity during mouse thymocyte development

Heat shock protein synthesis was examined in mouse thymocytes at three stages of development: early embryonic thymocytes, which are CD4^?CD8^?, adult thymocytes, which are primarily CD4⁺CD8⁺, and mature spleen T cells, which are CD4⁺CD8^? or CD4^?CD8⁺. After either a 41°C or 42°C heat shock, the synthesis of the maior heat-inducible protein (hsp68) was elevated during the first hour of recovery but then decreased abruptly in thymocytes from adult mice. In contrast, the synthesis of hsp68 continued for up to 4 h after heating embryonic mouse thymocytes or mature spleen T cells. The more rapid termination ofthe heat shock response in the adult thymocytes was not the result of eitherless heat damage or more rapid repair since the recovery of general protein synthesis was more severely delayed in these cells. As well, the double positive CD4⁺CD8⁺ cells were more sensitive to hyperthermia than either the double negative CD4^?CD8^? or single positive CD4⁺CD8^? or CD4^?CD8⁺ cells. Exposure of fetal thymus organ cultures to elevated temperature revealed that the double negative thymocytes were able to survive and differentiate normally following a heat shock treatment that was lethal for the double positive thymocytes. Exposure of thymocytes from adult mice to elevated temperatures induced apoptotic cell death. This was evident by the cleavage of DNA into oligonucleosome-sized fragments. Quantitation of the extent of DNA fragmentation and the number of apoptotic cells by flow cytometry demonstrated that the extent of apoptotic cell death was related to the severity of the heat stress. Double positive (CD4+CD8+) thymocytes are selected on the basis of their T-cell antigen receptor (TCR). Most of these cells are negatively selected and die within the thymus by an active process of cell deletion known as apoptosis. Restricting hsp synthesis in response to stress might be essential during developmental processes in which cell maturation is likely to result in death rather than functional differentiation. © 1993Wiley-Liss, Inc.     

Induced thermotolerance in bovine two-cell embryos and the role of heat shock protein 70 in embryonic development

Induced thermotolerance is a phenomenon whereby exposure to a mild heat shock can induce heat shock proteins (HSP) and other cellular changes to make cells more resistant to a subsequent, more severe heat shock. Given that the 2-cell bovine embryo is very sensitive to heat shock, but can also produce HSP70 in response to elevated temperature, experiments were conducted to test whether 2-cell embryos could be made to undergo induced thermotolerance. Another objective was to test the role of the heat-inducible form of heat shock protein 70 (HSP70i) in development and sensitivity of bovine embryos to heat shock. To test for induced thermotolerance, 2-cell bovine embryos were first exposed to a mild heat shock (40 degrees C for 1 hr, or 41 degrees C or 42 degrees C for 80 min), allowed to recover at 38.5 degrees C and 5% (v/v) CO2 for 2 hr, and then exposed to a severe heat shock (41 degrees C for 4.5, 6, or 12 hr). Regardless of the conditions, previous exposure to mild heat shock did not reduce the deleterious effect of heat shock on development of embryos to the blastocyst stage. The role of HSP70i in embryonic development was tested in two experiments by culturing embryos with a monoclonal antibody to the inducible form of HSP70. At both 38.5 degrees C and 41 degrees C, the proportion of 2-cell embryos that developed to blastocyst was reduced (P < 0.05) by addition of anti-HSP70i to the culture medium. In contrast, sensitivity to heat shock was not generally increased by addition of antibody. In conclusion, bovine 2-cell embryos appear incapable of induced thermotolerance. Lack of capacity for induced thermotolerance could explain in part the increased sensitivity of 2-cell embryos to heat shock as compared to embryos at later stages of development. Results also implicate a role for HSP70i in normal development of bovine embryos.     

Effect of heat shock,pretreatment and hsp70 copy number on wing development in Drosophila melanogaster

Naturally occurring heat shock (HS) during pupation induces abnormal wing development in Drosophila; we examined factors affecting the severity of this induction. The proportion of HS-surviving adults with abnormal wings varied with HS duration and intensity, and with the pupal age or stage at HS administration. Pretreatment (PT), mild hyperthermia delivered before HS, usually protected development against HS. Gradual heating resembling natural thermal regimes also protected wing development against thermal disruption. Because of the roles of the wings in flight and courtship and in view of natural thermal regimes that Drosophila experience, both HS-induction of wing abnormalities and its abatement by PT may have marked effects on Drosophila fitness in nature. Because PT is associated with expression of heat-inducible molecular chaperones such as Hsp70 in Drosophila, we compared thermal disruption of wing development among hsp70 mutants as well as among strains naturally varying in Hsp70 levels. Contrary to expectations, lines or strains with increased Hsp70 levels were no more resistant to HS-disruption of wing development than counterparts with lower Hsp70 levels. In fact, wing development was more resistant to HS in hsp70 deletion strains than control strains. We suggest that, while high Hsp70 levels may aid cells in surviving hyperthermia, high levels may also overly stimulate or inhibit numerous signalling pathways involved in cell proliferation, maturation and programmed death, resulting in developmental failure.     

Impact of heat shock on heat shock proteins expression, biological and commercial traits of Bombyx mori

We report the thermotolerance of new bivoltine silkworm, Bombyx mori strains NB₄D₂, KSO₁, NP₂, CSR₂ and CSR₄and differential expression of heat shock proteins at different instars. Different instars of silkworm larva were subjected to heat shock at 35°C, 40°C and 45°C for 2 hours followed by 2 hours recovery. Heat shock proteins were analyzed by SDS‐PAGE. The impact of heat shock on commercial traits of cocoons was analyzed by following different strategies in terms of acquired thermotolerance over control. Comparatively NP₂ exhibited better survivability than other strains. Resistance to heat shock was increased as larval development proceeds in the order of first instar > second instar > third instar > fourth instar > fifth instar in all silkworm strains. Expression of heat shock proteins varies in different instars. 90 kDa in the first, second and third instars, 84 kDa in the fourth instar and 84, 62, 60, 47 and 33 kDa heat shock proteins in fifth instar was observed in response to heat shock. Relative influence of heat shock on commercial traits that correspond to different stages was significant in all strains. In NB₄D₂, cocoon and shell weight significantly increased to 17.52% and 19.44% over control respectively. Heat shock proteins as molecular markers for evaluation and evolution of thermotolerant silkworm strains for tropics was discussed.     

Effect of heat shock on ribosome synthesis in Drosophila melanogaster.

In Drosophila tissue culture cells, the synthesis of ribosomal proteins was inhibited by a 1-h 37 degrees C heat shock. Ribosomal protein synthesis was repressed to a greater extent than that of most other proteins synthesized by these cells at 25 degrees C. After a 1-h heat shock, when the cells were returned to 25 degrees C, the ribosomal proteins were much slower than most other 25 degrees C proteins to return to pre-heat shock levels of synthesis. Relative to one another, all the ribosomal proteins were inhibited and later recovered to normal levels of synthesis at the same rate and to the same extent. Unlike the ribosomal proteins, the precursor to the large rRNAs was continually synthesized during heat shock, although at a slightly reduced level, but was not processed. It was rapidly degraded, with a half-life of approximately 16 min. Pre-heat shock levels of synthesis, stability, and correct processing were restored only when ribosomal protein synthesis returned to at least 50% of that seen in non-heat-shocked cells.     

Thermotolerance induced by heat shock in Chlorella

Thermotolerance in cultures of Chlorella zofingiensis was induced by heat shock treatment at supraoptimal temperatures (40and 45 °C for 30 min). Thermotolerance was assayed by two methods: the survival of the cells at 70 °C and the growth of diluted cultures at 35 and 45 °C. A culture without heat shock treatment was unable to grow at 45 °C. According to eletrophoretic analyses, the synthesis of proteins of 95, 73, 60, 43 and 27 kDa was induced by heat shock treatment. The large molecular weight proteins (95, 73, 60 and43 kDa) were present in non-heat treated cells, but the heat shock treatment increased their quantity in cells. The synthesis of a low molecular weight protein (27 kDa) was induced by heat shock treatment. The induced thermotolerance could be inhibited by the presence of an 80S ribosomal translation inhibitor, cycloheximide(CHI). The first 12 amino acid residues from the N-terminus of the27 kDa heat shock induced protein are Val-Glu-Trp-Try-Gly-Pro-Asn-Arg-Ala-Lys-Phe-Leu. This revised version was published online in August 2006 with corrections to the Cover Date.     

Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption

The mammalian small heat shock protein (sHSPs) family is comprised of 10 members and includes HSPB1, which is proposed to play an essential role in cellular physiology, acting as a molecular chaperone to regulate diverse cellular processes. Whilst differential roles for sHSPs are suggested for specific tissues, the relative contribution of individual sHSP family members in cellular and organ physiology remains unclear. To address the function of HSPB1 in vivo and determine its tissue-specific expression during development and in the adult, we generated knock-in mice where the coding sequence of hspb1 is replaced by a lacZ reporter gene. Hspb1 expression marks myogenic differentiation with specific expression first confined to developing cardiac muscles and the vascular system, and later in skeletal muscles with specific expression at advanced stages of myoblast differentiation. In the adult, hspb1 expression was observed in other tissues, such as stratified squamous epithelium of skin, oronasal cavity, tongue, esophagus, and uterine cervix but its expression was most prominent in the musculature. Interestingly, in cardiac muscle hsbp1 expression was down-regulated during the neonatal period and maintained to a relatively low steady-level throughout adulthood. Despite this widespread expression, hspb1-/- mice were viable and fertile with no apparent morphological abnormalities in tissues under physiological conditions. However, at the cellular level and under stress conditions (heat challenge), HSPB1 act synergistically with the stress-induced HSPA1 (HSP70) in thermotolerance development, protecting cells from apoptosis. Our data thus indicate a nonessential role for HSPB1 in embryonic development and for maintenance of tissues under physiological conditions, but also shows that it plays an important role by acting synergistically with other HSPs during stress conditions to exert cytoprotection and anti-apoptotic effects.     

Differential responses of bovine oocytes and preimplantation embryos to heat shock

The authors sought to determine whether developmental differences in the magnitude of embryonic mortality caused by heat stress in vivo are caused by changes in resistance of embryos to elevated temperature. In this regard, responses of oocytes, two-cell embryos, four- to eight-cell embryos, and compacted morulae to heat shock were compared. An additional goal was to define further the role of cumulus cells and glutathione in thermoprotection of oocytes. In experiment 1, heat shock (41°C for 12 hr) decreased the number of embryos developing to the blastocyst stage for two-cell (26% vs. 0%) and four- to eight-cell (25% vs. 10%) embryos but did not affect morulae (37% vs. 42%). In experiment 2, exposure of two-cell embryos to 41°C for 12 hr reduced the number of four- to eight-cell embryos present 24 hr after the end of heat shock (88% vs. 62%). In experiment 3, heat shock reduced the number of two-cell embryos developing to blastocyst (49% vs. 8%) but did not affect subsequent development of oocytes when heat shock occurred during the first 12 hr of maturation (46% vs. 41% development to blastocyst); membrane integrity was not altered. In experiment 4, oocytes were cultured with an inhibitor of glutathione synthesis, _DL-buthionine-[S,R]-sulfoximine (BSO), for 24 hr and exposed to 41°C for the first 12 hr of maturation. Percentages of blastocysts were 35% (39°C), 18% (41°C), 17% (39°C+BSO), and 11% (41°C+BSO). For experiment 5, oocytes were either denuded or left with cumulus intact and were then radiolabeled with [³⁵S]methionine and [³⁵S]cysteine at 39°C or 41°C for 12 hr. Exposure of oocytes to 41°C for 12 hr reduced overall synthesis of ³⁵S-labeled TCA-precipitable intracellular proteins (18,160 vs. 14,594 dpm/oocyte), whereas presence of cumulus increased synthesis (9,509 vs. 23,246). Analysis by two-dimensional SDS PAGE and fluorography revealed that heat shock protein 68 (HSP68) and two other putative heat shock proteins, P71 and P70, were synthesized by all oocytes regardless of treatment. Heat shock did not alter the synthesis of HSP68 or P71 but decreased amounts of newly synthesized P70. Cumulus cells increased synthesis of P71 and P70. Results indicate there is a biphasic change in resistance to elevations in temperature as oocytes mature, become fertilized, and develop. Resistance declines from the oocyte to the two-cell stage and then increases. Evidence suggests a role for cumulus cells in increasing HSP70 molecules and protein synthesis. Data also indicate a role for glutathione in oocyte function. Mol Reprod Dev 46:138–145, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of cold-induced heat shock protein expression in neonatal rat cardiomyocytes

Cardiac surgery is usually performed under conditions of cardioplegicischemic arrest. To protect the heart during the ischemic period, themyocardium is exposed to varying degrees of hypothermia. Althoughhyperthermia is known to induce the heat shock response, the moleculareffects of hypothermia on the myocardium have not been investigated. We havestudied the effect of hypothermia on the induction of heat shock proteins inprimary cultures of neonatal cardiomyocytes. Cold stress in cardiomyocytesinduced a 6 fold increase in the heat shock protein HSP70 as compared tocontrol. Increased HSP70 protein levels correlated with induction of HSP70mRNAs. Maximal levels of HSP70 protein appeared 4-6 h following recoveryfrom cold shock, indicating the transient nature of the response. Inductionof HSP25 mRNA was also observed in cold-shocked cardiomyocytes, even thoughincreased HSP25 protein levels were not detected. Our results indicate thathypothermia is capable of inducing the heat shock response in neonatalcardiomyocytes.     

Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein (HSP) genes (hsp70, hsc70, hsp90) were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus (Homoptera: Delphacidae), which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepi‐dopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock (40 °C) upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4°C did not change the expression levels of any hsp in either species.     

Cloning of heat shock protein genes from the brown planthopper,Nilaparvata lugens,and the small brown planthopper,Laodelphax striatellus,and their expression in relation to thermal stress

Three heat shock protein （HSP） genes （hsp70, hsc70, hsp90） were partially cloned from the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus （Homoptera： Delphacidae）, which are serious pests of the rice plant. Sequence comparisons at the deduced amino acid level showed that the three HSPs of planthoppers were most homologous to corresponding HSPs of dipteran and lepidopteran species. Identities of both heat shock cognate 70 and HSP90 were higher than HSP70 in both species. Identity of the HSP70 between the two planthopper species was only 81%, a value much lower than seen among fly and moth groups. Effects of heat and cold shocks were demonstrated on expression of the three hsp genes in the two planthopper species. Heat shock （40 ℃） upregulated the hsp90 level but did not change the hsc70 level in either the nymph and adult stages of either species. On the other hand, the hsp70 level was only upregulated in L. striatellus. This heat shock response was prompt and lasted only for 1 h after treatment. In contrast, cold shock at 4℃ did not change the expression levels of any hsp in either species.     

Basal cold but not heat tolerance constrains plasticity among Drosophila species (Diptera: Drosophilidae)

Thermal tolerance and its plasticity are important for understanding ectotherm responses to climate change. However, it is unclear whether plasticity is traded‐off at the expense of basal thermal tolerance and whether plasticity is subject to phylogenetic constraints. Here, we investigated associations between basal thermal tolerance and acute plasticity thereof in laboratory‐reared adult males of eighteen Drosophila species at low and high temperatures. We determined the high and low temperatures where 90% of flies are killed (ULT₉₀ and LLT₉₀, respectively) and also the magnitude of plasticity of acute thermal pretreatments (i.e. rapid cold‐ and heat‐hardening) using a standardized, species‐specific approach for the induction of hardening responses. Regression analyses of survival variation were conducted in ordinary and phylogenetically informed approaches. Low‐temperature pretreatments significantly improved LLT₉₀ in all species tested except for D. pseudoobscura, D. mojavensis and D. borealis. High‐temperature pretreatment only significantly increased ULT₉₀ in D. melanogaster, D. simulans, D. pseudoobscura and D. persimilis. LLT₉₀ was negatively correlated with low‐temperature plasticity even after phylogeny was accounted for. No correlations were found between ULT₉₀ and LLT₉₀ or between ULT₉₀ and rapid heat‐hardening (RHH) in ordinary regression approaches. However, after phylogenetic adjustment, there was a positive correlation between ULT₉₀ and RHH. These results suggest a trade‐off between basal low‐temperature tolerance and acute low‐temperature plasticity, but at high temperatures, increased basal tolerance was accompanied by increased plasticity. Furthermore, high‐ and low‐temperature tolerances and their plasticity are clearly decoupled. These results are of broad significance to understanding how organisms respond to changes in habitat temperature and the degree to which they can adjust thermal sensitivity.