Differences in egg thermotolerance between tropical and temperate populations of the migratory locust Locusta migratoria (Orthoptera: Acridiidae)

The migratory locust Locusta migratoria L., which is widely distributed throughout the world, exhibits within- and between-population variation in cold tolerance. To understand physiological adaptation in populations, we studied the genetic basis of thermotolerance in Hainan (tropical) and Liaoning (temperate) populations and measured expression of Hsp70 and Hsp90 mRNA in both populations at low (0 degrees C) and high temperatures (40 degrees C). Phenotypic variation of thermotolerance is heritable. Heritable characteristics differed among different stages of locust egg development, as well as among different measures of thermotolerance. Nuclear genetic factors, rather than cytoplasmic factors, contribute to differences in cold tolerance between the tropical and temperate populations of the migratory locust; for heat tolerance, maternal effects were involved in three stages of egg development. Expression of Hsp90 mRNA was induced in temperate population after heat shock (40 degrees C x 12h), whereas expression of Hsp70 and 90 was induced in tropical population after cold shock (0 degrees C x 12h). We suggest that thermotolerance of locust eggs has a complex genetic basis and heat shock proteins may be involved in differences of thermotolerance between locust populations.     

Molecular and biochemical modulation of heat shock protein 20 (Hsp20) gene by temperature stress and hydrogen peroxide (H₂O₂) in the monogonont rotifer, Brachionus sp

The monogonont rotifer, Brachionus sp. has been regarded as a potential model for reproductive physiology, evolution, and environmental genomics. To uncover the role of the heat shock protein upon temperature stress and hydrogen peroxide (H?O?) exposure, we cloned heat shock protein 20 (Hsp20) and determined its modulatory response under different temperatures and H?O? concentrations. Under different temperature stresses (10 °C and 37 °C), the rotifer Brachionus sp. Hsp20 (Br-Hsp20) gene was highly expressed over time, and reached the maximum level 90 min after exposure, indicating that Br-Hsp20 gene would be involved in the chaperoning process to protect proteins at both low and high temperatures. To test the ability of thermotolerance of the recombinant Br-Hsp20-containing transformed Escherichia coli, we expressed the recombinant Br-Hsp20 protein with 1mM IPTG for 18 h at 30 °C, exposed them at 54 °C with time course (10 to 60 min), and measured cell survival. In this elevated temperature shock (54 °C), the cell survival was significantly higher at the Br-Hsp20 transformed E. coli, compared to the control (vector only). To analyze the modulatory effect of Br-Hsp20 gene on oxidative stress, we initially exposed 0.1 mM H?O? over time and measured antioxidant enzyme activities along with the expression level of Br-Hsp20 mRNA. Upon H?O? exposure, Br-Hsp20 gene was time-dependently upregulated and glutathione peroxidase (GPx), glutathione S-transferase (GST), and glutathione reductase (GR) activities were also elevated at the 12h-exposed group in a dose-dependent manner, indicating that the Br-Hsp20 gene would be an important gene in response to oxidative and temperature stress. Here, we demonstrated the role of the Hsp20 gene in the rotifer, Brachionus sp. providing a better understanding of the ecophysiology at environmental stress in this species.     

Human mutation in the anti-apoptotic heat shock protein 20 abrogates its cardioprotective effects

The small heat shock protein Hsp20 protects cardiomyocytes against apoptosis, and phosphorylation at its Ser16 site enhances its cardioprotection. To determine whether genetic variants exist in human Hsp20, which may modify these beneficial effects, we sequenced the coding region of the Hsp20 gene in 1347 patients suffering from dilated cardiomyopathy and 744 subjects with no heart disease. We identified a C59T substitution in the human Hsp20 gene in one patient and three individuals without heart disease. All subjects were heterozygous for this mutation, which changes a fully conserved proline residue into leucine at position 20 (P20L), resulting in secondary structural alterations. To examine the potential functional significance of the P20L-Hsp20 human variant, adult rat cardiomyocytes were infected with Ad.GFP (where Ad is adenovirus and GFP is green fluorescent protein), Ad.WT-Hsp20 (where WT is wild-type), and Ad.P20L-Hsp20 and subjected to simulated ischemia/reperfusion injury. Expression of WT-Hsp20 resulted in significant attenuation of apoptosis compared with the GFP control. However, the P20L-Hsp20 mutant showed no protection against apoptosis, assessed by Hoechst staining and DNA fragmentation. The loss of cardioprotection by the mutant Hsp20 was associated with its diminished phosphorylation at Ser16 compared with WT-Hsp20. Furthermore, maximal stimulation of cardiomyocytes with isoproterenol or protein kinase A-mediated phosphorylation in vitro confirmed the impaired ability of the mutant Hsp20 to become phosphorylated at Ser16. In conclusion, we have identified a P20L substitution in human Hsp20, which is associated with diminished phosphorylation at Ser16 and complete abrogation of the Hsp20 cardioprotective effects which may adversely affect the ability of human carriers to cope with cellular stress.     

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.     

Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 °C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.     

Hsp20 functions as a novel cardiokine in promoting angiogenesis via activation of VEGFR2

Heat shock proteins (Hsps) are well appreciated as intrinsic protectors of cardiomyocytes against numerous stresses. Recent studies have indicated that Hsp20 (HspB6), a small heat shock protein, was increased in blood from cardiomyopathic hamsters. However, the exact source of the increased circulating Hsp20 and its potential role remain obscure. In this study, we observed that the circulating Hsp20 was increased in a transgenic mouse model with cardiac-specific overexpression of Hsp20, compared with wild-type mice, suggesting its origin from cardiomyocytes. Consistently, culture media harvested from Hsp20-overexpressing cardiomyocytes by Ad.Hsp20 infection contained an increased amount of Hsp20, compared to control media. Furthermore, we identified that Hsp20 was secreted through exosomes, independent of the endoplasmic reticulum-Golgi pathway. To investigate whether extracellular Hsp20 promotes angiogenesis, we treated human umbilical vein endothelial cells (HUVECs) with recombinant human Hsp20 protein, and observed that Hsp20 dose-dependently promoted HUVEC proliferation, migration and tube formation. Moreover, a protein binding assay and immunostaining revealed an interaction between Hsp20 and VEGFR2. Accordingly, stimulatory effects of Hsp20 on HUVECs were blocked by a VEGFR2 neutralizing antibody and CBO-P11 (a VEGFR inhibitor). These in vitro data are consistent with the in vivo findings that capillary density was significantly enhanced in Hsp20-overexpressing hearts, compared to non-transgenic hearts. Collectively, our findings demonstrate that Hsp20 serves as a novel cardiokine in regulating myocardial angiogenesis through activation of the VEGFR signaling cascade.     

Effects of hyperthermia on spermatogenesis, apoptosis, gene expression, and fertility in adult male mice.

Testicular heat shock was used to characterize cellular and molecular mechanisms involved in male fertility. This model is relevant because heat shock proteins (HSPs) are required for spermatogenesis and also protect cells from environmental hazards such as heat, radiation, and chemicals. Cellular and molecular methods were used to characterize effects of testicular heat shock (43 degrees C for 20 min) at different times posttreatment. Mating studies confirmed conclusions, based on histopathology, that spermatocytes are the most susceptible cell type. Apoptosis in spermatocytes was confirmed by TUNEL, and was temporally correlated with the expression of stress-inducible Hsp70-1 and Hsp70-3 proteins in spermatocytes. To further characterize gene expression networks associated with heat shock-induced effects, we used DNA microarrays to interrogate the expression of 2208 genes and thousands more expression sequence tags expressed in mouse testis. Of these genes, 27 were up-regulated and 151 were down-regulated after heat shock. Array data were concordant with the disruption of meiotic spermatogenesis, the heat-induced expression of HSPs, and an increase in apoptotic spermatocytes. Furthermore, array data indicated increased expression of four additional non-HSP stress response genes, and eight cell-adhesion, signaling, and signal-transduction genes. Decreased expression was recorded for 10 DNA repair and recombination genes; 9 protein synthesis, folding, and targeting genes; 9 cell cycle genes; 5 apoptosis genes; and 4 glutathione metabolism genes. Thus, the array data identify numerous candidate genes for further analysis in the heat-shocked testis model, and suggest multiple possible mechanisms for heat shock-induced infertility.     

Characterization of goldfish heat shock protein-30 induced upon severe heat shock in cultured cells

Temperature-dependent changes of growth rate and protein components were investigated for primary cultured cells derived from goldfish caudal fin. When the culture temperature was shifted from 20 degrees C to 35 degrees C and 40 degrees C, the growth rate was increased at 35 degrees C as compared with that at 20 degrees C, but no cell growth was observed at 40 degrees C. The differential scanning calorimetry demonstrated the onset of the endothermic reaction for goldfish cellular components at 40 degrees C. Therefore, the temperature shift to 40 degrees C was found to be of severe heat shock for goldfish cultured cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that, although expression of 70-kDa components was slightly induced at 35 degrees C, the temperature shift to 40 degrees C markedly induced the expression of the 30-kDa component in addition to that of 70-kDa component. The N-terminal amino acid sequencing identified the 30- and 70-kDa components to be heat shock protein (Hsp)-30 and Hsp70, respectively. Northern blot analysis revealed that the enhanced Hsp30 messenger ribonucleic acid (mRNA) levels were only observed at 40 degrees C, whereas Hsp70 mRNA was slightly accumulated at 35 degrees C. These results indicated that Hsp30 might have important functions under severe heat stress condition.     

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.     

Characterization of Hsp70 gene in Chironomus riparius: expression in response to endocrine disrupting pollutants as a marker of ecotoxicological stress

We characterized the Hsp70 cDNA in Chironomus riparius and evaluated its expression profile under different environmental stressors. It is highly conserved, at both DNA and protein levels, displaying many of the hallmarks of Hsps and sharing 80-96% of overall amino acid identities with homologous sequences from other diptera. The changes are mainly concentrated in the C-terminal domain of the protein. Phylogenetic analysis was consistent with the known classification of insects. The Hsp70 gene was located by in situ hybridization in region III-3A at the third polytene chromosome, a locus activated upon heat shock as shown by RNA pol II binding. As C. riparius is widely used in aquatic ecotoxicology testing, we studied Hsp70 gene induction in fourth instar aquatic larvae submitted to heat shock and selected environmental pollutants classified as potential endocrine disruptors. RT-PCR analysis showed that Hsp70 mRNA levels increased significantly (p<0.05) after short-term acute exposures to a temperature shift (HS), cadmium chloride (Cd), butyl benzyl phthalate (BBP), diethylhexyl phthalate (DEHP), bisphenol A (BPA), 4-nonylphenol (NP) and ethinylestradiol (EE). However, neither pentachlorophenol (PCP) nor tributyltin (TBTO) treatments were able to activate the Hsp70 gene. The cognate form, Hsc70, was also analysed and, unlike Hsp70, was not altered by any of the different treatments assayed. Moreover, at the times tested, there was no significant mortality of the larvae. The rapid upregulation of the Hsp70 gene suggests that it is sensitive and selective for different environmental pollutants, and could be used as an early molecular endpoint in ecotoxicological studies.     

Cell type–specific variations in the induction of hsp70 in human leukocytes by feverlike whole body hyperthermia

Fever has been associated with shortened duration and improved survival in infectious disease. The mechanism of this beneficial response is still poorly understood. The heat-inducible 70-kDa heat shock protein (Hsp70) has been associated with protection of leukocytes against the cytotoxicity of inflammatory mediators and with improved survival of severe infections. This study characterizes the induction of Hsp70 by feverlike temperatures in human leukocytes in vitro and in vivo. Using flow cytometry, Hsp70 expression was determined in whole blood samples. This approach eliminated cell isolation procedures that would greatly affect the results. Heat treatment of whole blood in vitro for 2 hours at different temperatures revealed that Hsp70 expression depends on temperature and cell type; up to 41 degrees C, Hsp70 increased only slightly in lymphocytes and polymorphonuclear leukocytes. However, in monocytes a strong induction was already seen at 39 degrees C, and Hsp70 levels at 41 degrees C were 10-fold higher than in the 37 degrees C control. To be as close as possible to the physiological situation during fever, we immersed healthy volunteers in a hot water bath, inducing whole body hyperthermia (39 degrees C), and measured leukocyte Hsp70 expression. Hsp70 was induced in all leukocytes with comparable but less pronounced cell type-specific variations as observed in vitro. Thus, a systemic increase of body temperature as triggered by fever stimulates Hsp70 expression in peripheral leukocytes, especially in monocytes. This fever-induced Hsp70 expression may protect monocytes when confronted with cytotoxic inflammatory mediators, thereby improving the course of the disease.     

Babesia divergens: identification and characterization of BdHSP-20, a small heat shock protein

This study describes the identification and characterization of the Babesia divergens α-crystallin/small heat shock protein 20 (BdHSP-20). BdHSP-20 was recognized by the DG7 monoclonal antibody (DG7 mAb) originally produced by Precigout et al. [Precigout, E., Valentin, A., Carcy, B., Gorenflot, A., Nakamura, K., Aikawa, M., Schrevel, J. 1993. Babesia divergens: characterization of a 17-kDa merozoite membrane protein. Experimental Parasitology 77, 425-434] against B. divergens merozoites. We used DG7 mAb to immunoscreen a B. divergens cDNA library to clone the gene encoding the small heat shock protein. Bdhsp-20 is a single copy gene interrupted by one intron. The deduced gene product (BdHSP-20) clearly belongs to the α-crystallin family and shows significant homology to Babesia bovis, Plasmodiumfalciparum and Toxoplasma gondii sHSPs, with the highest degree of sequence identity around the catalytic domain. Nutritient stress (serum depletion) treatment of the parasites induced the upregulation of BdHSP-20 gene expression observed by semi-quantitative PCR and immunoprecipitation. This regulation pattern suggests that BdHSP-20 could probably be of importance for parasite survival in the case of environmental stress. BdHSP-20 has previously been shown to be highly conserved among different strains and antibodies against the protein drastically reduce parasitemia in vitro.