Expression and localization of Hsps in the heart and blood vessel of heat-stressed broilers

The objective of this study was to investigate the kinetics of Hsp60, Hsp70, Hsp90 protein, and messenger RNA (mRNA) expression levels and to correlate these heat shock protein (Hsp) levels with tissue damage resulting from exposure to high temperatures for varying amounts of time. One hundred broilers were heat-stressed for 0, 2, 3, 5, and 10 h, respectively, by rapidly increasing the ambient temperature from 22 +/- 1 degrees C to 37 +/- 1 degrees C. Obvious elevations of plasma creatine kinase indicate damage to myocardial cells after heat stress. Hsp70 and Hsp90, and their corresponding mRNAs in the heart tissue of heat-stressed broilers, elevated significantly after 2 h of heat exposure and decreased quickly with continued heat stress. However, the levels of hsp60 mRNA in the heart of heat-stressed broilers increased sharply (P < 0.01) at 2 h of heat stress but then decreased quickly after 3 h, while the level of Hsp60 protein in the heart increased (P < 0.01) at 2 h of heat stress and maintained a high level throughout heat exposure. The results indicate that the elevation of the three Hsps, especially Hsp60 in heart, may be important markers at the beginning of heat stress and act as protective proteins in adverse environments. The reduction of Hsp signals in the cytoplasm of myocardial cells implies that myocardial cell lesions may have an adverse impact on the function of Hsps during heat stress. Meanwhile, the localization of Hsp70 in blood vessels of broiler hearts suggests another possible mechanism for protection of the heart after heat exposure.     

Agonistic encounters and cellular angst: social interactions induce heat shock proteins in juvenile salmonid fish

Juvenile salmonid fish readily form dominance hierarchies when faced with limited resources. While these social interactions may result in profound behavioural and physiological stress, it is unknown if this social stress is evident at the level of the cellular stress response—specifically, the induction of stress or heat shock proteins (Hsps). Thus, the goal of our study was to determine if Hsps are induced during hierarchy formation in juvenile rainbow trout (Oncorhynchus mykiss). To this end, we measured levels of three Hsps, Hsp70, Hsc (heat shock cognate)70 and Hsp90 in the white muscle, liver and brain of trout that had been interacting for 36 h, 72 h or 6 days. Our data indicate that Hsps are induced in both dominant and subordinate fish in a time- and tissue-specific manner. In further mechanistic experiments on fasted and cortisol-treated fish, we demonstrated that high plasma cortisol does not affect Hsp induction in trout white muscle or liver, but both conditions may be part of the mechanism for Hsp induction with social stress in the brain. We conclude that the behavioural and physiological stress experienced by juvenile rainbow trout in dominance hierarchies can be extended to the induction of Hsps.     

Expression and distribution of heat shock proteins in the heart of transported pigs

The expression and localization of four heat shock proteins (Hsp70, Hsp86, Hsp90, and Hsp27) were shown in the heart tissue of pigs transported for 6 h. Immunostaining detected the consistent presence of all Hsps in the pig myocardial cells under both transported and normal housing conditions. Immunohistochemical analysis revealed predominance of Hsp70 (significantly highest levels) and Hsp27 in the cytoplasm of myocardial cells. Hsp90 and Hsp86 were expressed both in the cytoplasm and in the nucleus, preferentially in the cytoplasm, of the myocardial cells. In view of their abundant and uniform distributions in the myocardial cells, the expression and distribution patterns of all detected Hsps within the myocardial cells, mostly limited to the cytoplasm, could be related to their chaperone function for cells with important special activities in this study. The identification of all four Hsps in the blood vessel endothelial cells possibly implies that endothelial cells react to ischemia and hypoxia by expressing Hsps. Immunoblot findings suggest that the level of all Hsps decreased in response to stress due to a 6 h journey. The decrease in Hsp levels in the myocardial cells may indicate that the transport stress may have overcharged the repair mechanisms of the cells. Whether this distinct depletion of Hsps contributes to an increased susceptibility to acute heart failure and the sudden death syndrome in transported pigs should be elucidated in future experiments.     

Heat-shock proteins are induced in unstressed leaves of Nicotiana attenuata (Solanaceae) when distant leaves are stressed

In Nicotiana attenuata, systemic induction of heat-shock proteins (Hsps) was detected in response to the treatment of single leaves by either heat shock, mechanical damage, or exogenous application of methyl jasmonate (MJ). All treatments increased the abundance of members of the 70-kD Hsp (Hsp70) family and induced synthesis of one or more of the small Hsps (sHsp) (16-23 kD) in both treated and untreated leaves. These results provide the first evidence that Hsps can be systemically induced in plants and suggest that systemic induction of Hsps may be important in pre-adapting leaves to stress.     

Human esophageal microvascular endothelial cells respond to acidic pH stress by PI3K/AKT and p38 MAPK-regulated induction of Hsp70 and Hsp27

The heat shock response maintains cellular homeostasis following sublethal injury. Heat shock proteins (Hsps) are induced by thermal, oxyradical, and inflammatory stress, and they chaperone denatured intracellular proteins. Hsps also chaperone signal transduction proteins, modulating signaling cascades during repeated stress. Gastroesophageal reflux disease (GERD) affects 7% of the US population, and it is linked to prolonged esophageal acid exposure. GERD is characterized by enhanced and selective leukocyte recruitment from esophageal microvasculature, implying activation of microvascular endothelium. We investigated whether phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK regulate Hsp induction in primary cultures of human esophageal microvascular endothelial cells (HEMEC) in response to acid exposure (pH 4.5). Inhibitors of signaling pathways were used to define the contribution of PI3K/Akt and MAPKs in the heat shock response and following acid exposure. Acid significantly enhanced phosphorylation of Akt and MAPKs in HEMEC as well as inducing Hsp27 and Hsp70. The PI3K inhibitor LY-294002, and Akt small interfering RNA inhibited Akt activation and Hsp70 expression in HEMEC. The p38 MAPK inhibitor (SB-203580) and p38 MAPK siRNA blocked Hsp27 and Hsp70 mRNA induction, suggesting a role for MAPKs in the HEMEC heat shock response. Thus acidic pH exposure protects HEMEC through induction of Hsps and activation of MAPK and PI3 kinase pathway. Acidic exposure increased HEMEC expression of VCAM-1 protein, but not ICAM-1, which may contribute to selective leukocyte (i.e., eosinophil) recruitment in esophagitis. Activation of esophageal endothelial cells exposed to acidic refluxate may contribute to GERD in the setting of a disturbed mucosal squamous epithelial barrier (i.e., erosive esophagitis, peptic ulceration). esophagus; esophagitis; gastroesophageal reflux disease; microvasculature; phosphatidylinositol 3-kinase/Akt; VCAM-1     

70-kDa heat shock protein expression in cultured rat astrocytes after hypoxia: regulatory effect of almitrine

Induction of heat shock proteins (Hsps), especially the 70-kDa family, is well observed in nervous tissues in response to various stressful conditions. By using rat astrocytes in primary culture, the expression of the inducible (Hsp70) and the constitutive (Hsc70) 70-kDa Hsps immunoreactivity of cells exposed to hypoxic conditions has been investigated. We observed that exposure of astroglial cells to an hypoxic-normoxic sequence induces a significant decrease of Hsc70 immunoreactivity. The presence of the heat inducible stress protein Hsp70 is never observed in hypoxic cells not in control. Hsc 70 lowering is associated with ultrastructural alterations characterized by mitochondria swelling, formation of vacuoles and accumulation of dense material in the cell cytoplasm. The effects of addition of almitrine to the culture medium before and during hypoxia on Hsps immunoreactivity have been examined. The presence of the drug prevents the decrease of Hsc 70 immunoreactivity induced by hypoxia. Furthermore, some ultrastructural improvement is observed in astroglial cells treated with almitrine suggesting some protecting role of Hsc70 on cell damage induced by hypoxia.     

Localization and Expression of Hsp27 and αB-Crystallin in Rat Primary Myocardial Cells during Heat Stress In Vitro

Neonatal rat primary myocardial cells were subjected to heat stress in vitro, as a model for investigating the distribution and expression of Hsp27 and αB-crystallin. After exposure to heat stress at 42°C for different durations, the activities of enzymes expressed during cell damage increased in the supernatant of the heat-stressed myocardial cells from 10 min, and the pathological lesions were characterized by karyopyknosis and acute degeneration. Thus, cell damage was induced at the onset of heat stress. Immunofluorescence analysis showed stronger positive signals for both Hsp27 and αB-crystallin from 10 min to 240 min of exposure compared to the control cells. According to the Western blotting results, during the 480 min of heat stress, no significant variation was found in Hsp27 and αB-crystallin expression; however, significant differences were found in the induction of their corresponding mRNAs. The expression of these small heat shock proteins (sHsps) was probably delayed or overtaxed due to the rapid consumption of sHsps in myocardial cells at the onset of heat stress. Our findings indicate that Hsp27 and αB-crystallin do play a role in the response of cardiac cells to heat stress, but the details of their function remain to be investigated.     

Mild heat stress at a young age in<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> leads to increased Hsp70 synthesis after stress exposure later in life

In a number of animal species it has been shown that exposure to low levels of stress at a young age has a positive effect on stress resistance later in life, and on longevity. The positive effects have been attributed to the activation of defence/cleaning systems (heat shock proteins (Hsps), antioxidases, DNA repair) or to effects of a changed metabolic rate, or both. We investigated the effect of mild stress exposures early in life on Hsp70 synthesis after a harder stress exposure later in life in five isofemale lines ofDrosophila melanogaster. Female flies were either exposed to repeated bouts of mild heat stress (3 h at 34‡C) at a young age (days 2, 4 and 6 post-eclosion) or held under standard laboratory conditions. At 16 and 32 days of adult age, respectively, flies were exposed to a high temperature treatment known to induce Hsp70 in the investigated species (1 h at 37‡C). Thereafter, the inducible Hsp70 levels were measured. Our data show a tendency towards increased Hsp70 synthesis with increased age for both ’mild stress’ and ’no stress’ flies. Moreover, the results show that flies exposed to mild stress at a young age synthesized more Hsp70 upon induction, compared to control flies, and that this difference was accentuated at 32 days compared to 16 days of age. Thus, bouts of mild heat stress at a young age impact on the physiological stress response system later in life. This may be caused by an increased ability to react to future stresses. Alternatively, the mild stress exposure at a young age may actually have caused cellular damages increasing the need for Hsp70 levels after stress exposure later in life. The importance of an Hsp70 upregulation (throughout life) in explaining the phenomenon of hormesis is discussed, together with alternative hypotheses, and suggestions for further studies.     

Hsp70 from Cyprinion macrostomus macrostomus and Garra rufa obtuse: Stability and stability-dependent activity

Two fish species, Cyprinion macrostomus macrostomus and Garra rufa obtuse, tolerate adverse conditions in the Kangal hot springs and cope with multiple stressors such as food deprivation, extreme temperature, toxins, protein degradation, hypoxia, and microbial damage. These fish have evolved strategies to counteract the stressors including the induction of heat shock proteins (Hsps). Hsps play an essential role in maintaining cellular homeostasis, and one of the key proteins in the mechanism is Hsp70. Hsp70 itself is exposed to the same stressors as all other proteins, and, hence, the stability of Hsp70 was investigated. For this purpose, Hsp70 ATPase activity was determined at different urea concentrations. It was found that the protein maintains considerable ATP hydrolysis activity at higher denaturant conditions. Temperature effects on the substrate peptide binding showed that Hsp70s bind prominently at elevated temperatures. Furthermore, temperature effects on Hsp70 aggregation indicated that the presence of nucleotides decreases the aggregation process. The present work has determined the stability and activity of cmHsp70 and grHsp70 themselves under extreme conditions. The stability of the Hsp70 proteins maintains substrate proteins in the native state, which may aid in the adaptation of the fish species to the hot spring environment.     

The role of stress proteins in responses of a montane willow leaf beetle to environmental temperature variation

The heat shock response is a critical mechanism by which organisms buffer effects of variable and unpredictable environmental temperatures. Upregulation of heat shock proteins (Hsps) increases survival after exposure to stressful conditions in nature, although benefits of Hsp expression are often balanced by costs to growth and reproductive success. Hsp-assisted folding of variant polypeptides may prevent development of unfit phenotypes; thus, some differences in Hsp expression among natural populations of ectotherms may be due to interactions between enzyme variants (allozymes) and Hsps. In the Sierra willow leaf beetle Chrysomela aeneicollis, which lives in highly variable thermal habitats at the southern edge of their range in the Eastern Sierra Nevada, California, allele frequencies at the enzyme locus phosphoglucose isomerase (PGI) vary across a climatic latitudinal gradient. PGI allozymes differ in kinetic properties, and expression of a 70 kDa Hsp differs between populations, along elevation gradients, and among PGI genotypes. Differences in Hsp 70 expression among PGI genotypes correspond to differences in thermal tolerance and traits important for reproductive success, such as running speed, survival and fecundity. Thus, differential Hsp expression among genotypes may allow functionally important genetic variation to persist, allowing populations to respond effectively to environmental change.     

Heat shock protein 70 is translocated to lipid droplets in rat adipocytes upon heat stimulation

In mammalian cells, lipid storage droplets contain a triacylglycerol and cholesterol ester core surrounded by a phospholipid monolayer into which a number of proteins are imbedded. These proteins are thought to be involved in modulating the formation and metabolic functions of the lipid droplet. In this study, we show that heat stress upregulates several heat shock proteins (Hsps), including Hsp27, Hsp60, Hsp70, Hsp90, and Grp78, in primary and differentiated adipocytes. Immunostaining and immunoblotting data indicate that among the Hsps examined, only Hsp70 is induced to redirect to the lipid droplet surface in heat-stressed adipocytes. The thermal induction of Hsp70 translocation to lipid droplet does not typically happen in a temperature- or time-dependent manner and occurs abruptly at 30-40 min and rapidly achieves a steady state within 60 min after 40 degrees C stress of adipocytes. Though Hsp70 is co-localized with perilipin on the lipid droplets in stressed adipocytes, immunoprecipitation experiments suggest that Hsp70 does not directly interact with perilipin. Alkaline treatments indicate that Hsp70 associates with the droplet surface through non-hydrophobic interactions. We speculate that Hsp70 might noncovalently associate with monolayer microdomains of the lipid droplet in a manner similar to its interaction with lipid bilayer moieties composed of specific fatty acids. As an acute and specific cellular response to the heat stimulation, accumulation of Hsp70 on adipocytes lipid droplets might be involved in stabilizing the droplet monolayer, transferring nascent proteins to the lipid droplets, or chaperoning denatured proteins on the droplet for subsequent refolding.     

Quantitative evidence that both Hsc70 and Hsp70 contribute to thermal adaptation in hybrids of the livebearing fishes Poeciliopsis

The 70-kilodalton heat shock protein family is composed of both environmentally inducible (Hsp) and constitutively expressed (Hsc) family members. While the role of the constitutively expressed stress proteins in thermotolerance is largely unknown, de novo expression stress proteins in response to elevated temperatures has been associated with increased thermotolerance in many cell lines, developing embryos and adult organisms. Distinct, hemiclonal hybrids between the livebearing fish species Poeciliopsis monacha and P. lucida varied in their abilities to survive temperature stress, with survival being greatest when rates of temperature increase to 40°C were slowest and when P. monacha genomes were combined with a sympatric P. lucida genome. Quantification of Hsp70 under heat shock conditions and Hsc70 under normal physiological conditions indicated that variation in survival among hemiclones was best explained by the combined effects of these two proteins. Similar complex interactions between maternal and paternal genomes and rate of temperature increase were found to underline patterns of survival, Hsp70 accumulation and Hsc70 abundance. These data suggest that the relationship between Hsps and thermotolerance is more intricate than previously thought and that Hsps contribute to thermal adaptation in these fishes through genetic interactions specific to particular environments.     

Role of HSF activation for resistance to heat, cold and high-temperature knock-down

Regulation of heat shock proteins (Hsps) by the heat shock factor (HSF) and the importance of these proteins for resistance to heat stress is well documented. Less characterized is the importance of Hsps for cold stress resistance although Hsp70 is known to be induced following long-term cold exposure in Drosophila melanogaster. In this study, a temperature-sensitive HSF mutant line was used to investigate the role of HSF activation following heat hardening, rapid cold hardening (RCH) and long-term cold acclimation (LTCA) on heat and cold resistance, and this was correlated with Hsp70 expression. In addition, the effect of HSF activation on high-temperature knock-down resistance was evaluated. We found a significantly decreased HSF activation in the mutant line as compared to a corresponding control line following heat hardening, and this was correlated with decreased heat resistance of the mutant line. However, we did not find this difference in HSF activity to be important for resistance to cold stress or high-temperature knock-down. The findings indicate that induction of stress genes regulated by HSF, such as Hsps, although occurring following LTCA, are not of major importance for cold stress resistance and neither for RCH nor high-temperature knock-down resistance in D. melanogaster.     

Association of plasma antibodies against the inducible Hsp70 with hypertension and harsh working conditions

Autoantibodies against certain stress or heat shock proteins (Hsps) may play a role in the pathogenesis and/ or prognosis of some diseases. Using immunoblotting with human recombinant Hsps and univariate and multivariate logistic regression models, we have investigated the presence of antibodies against Hsp70, the inducible member of the 70-kDa family of heat shock proteins, and analyzed its possible association with hypertension and working conditions. Plasma and serum were collected from 764 steel mill workers from 6 work sites exposed to (1) severe noise; (2) severe noise and dust; (3) noise, dust, and heat; (4) noise and heat; (5) severe noise and heat; and (6) office conditions (control). Workers with prolonged exposure to stresses such as noise, dust, and high temperature and a combination of these in the workplace had a high incidence (26.6% to 40.2%) of antibodies to Hsp70 compared to the lowest incidence (18.6%) of antibodies to Hsp70 in the control group of office workers. Moreover, there was a statistical association of antibodies against Hsp70 with hypertension. The statistical correlation between the presence of antibodies to Hsp70 and hypertension is higher in the group of workers with blood pressure of 160/95 mmHg than in the 140/90-mmHg group after excluding possible effects of the workplace stresses. These results suggest that harsh workplace conditions can increase the production of antibodies against Hsp70 and that the presence of antibodies to this stress protein may be associated with hypertension. The precise mechanism for the elevation of antibodies against Hsps by environmental and workplace stresses and their relation to hypertension remains to be established.     

Flow cytometry is a rapid and reliable method for evaluating heat shock protein 70 expression in human monocytes

The increasing interest in stress/heat shock proteins (Hsps) as markers of exposure to environmental stress or disease requires an easily applicable method for Hsp determination in peripheral blood cells. Of these cells, monocytes preferentially express Hsps upon stress. An appropriate fixation/permeabilization procedure was developed, combined with immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Higher relative fluorescence intensity was observed in cells exposed to heat shock (HS), reflecting a higher expression of Hsp70 in these cells as compared with cells kept at 37°C. The heat-inducible increased Hsp70 expression was temperature- and time-dependent. Expression of Hsp70 was not uniform within the monocyte population, indicating the presence of subpopulations expressing variable levels of Hsp70 in response to HS. Simultaneous measurements of intracellular Hsp70 and membrane CD14 expression revealed that the higher Hsp70 inducibility coincided with the higher CD14 expression. Comparisons performed with biometabolic labelling, Western blotting, immunofluorescence and immunoperoxidase microscopic analysis, showed a high concordance between these different methods; however, cytometry was more sensitive for Hsp70 detection than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited for the determination of protein levels in individual cells from an heterogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.     

棉花粉蚧热休克蛋白基因的鉴定

热休克蛋白(heat shock proteins,Hsps)是生物体或细胞受到热胁迫后新合成的一类遗传上高度保守的蛋白,在昆虫应对外界环境因子胁迫时起着重要作用。为了系统研究棉花粉蚧Phenacoccus solenopsis Hsp基因家族,对棉花粉蚧转录组基因注释信息进行分析、获得目标序列,并应用NCBI上Blast X等软件进行比对、共鉴定出24条热激蛋白(Hsp)基因,包括3个Hsp90、8个Hsp70、2个Hsp60和11个s Hsp(small heat shock protein,s Hsp)基因。对棉花粉蚧与模式昆虫家蚕Bombyx mori、黑腹果蝇Drosophila melanogaster、赤拟谷盗Tribolium castaneum系统进化关系分析显示,昆虫的小分子量热休克蛋白s Hsp具有很强的种属特异性,Hsp70家族的保守性比s Hsp强。棉花粉蚧热激蛋白基因的鉴定为深入研究该虫Hsp与生长发育、抗逆境的相互关系奠定了基础。     

Functional stabilization of Kv1.5 protein by Hsp70 in mammalian cell lines

The aim of this study was to elucidate the mechanisms for regulations of cardiac Kv1.5 channel expression. We particularly focused on the role of heat shock proteins (Hsps). We tested the effects of Hsps on the stability of Kv1.5 channels using biochemical and electrophysiological techniques: co-expression of Kv1.5 and Hsp family proteins in mammalian cell lines, followed by Western blotting, immunoprecipitation, pulse-chase analysis, immunofluorescence and whole-cell patch clamp. Hsp70 and heat shock factor 1 increased the expression of Kv1.5 protein in HeLa and COS7 cells, whereas either Hsp40, 27 or 90 did not. Hsp70 prolonged the half-life of Kv1.5 protein. Hsp70 was co-immunoprecipitated and co-localized with Kv1.5-FLAG. Hsp70 significantly increased the immunoreactivity of Kv1.5 in the endoplasmic reticulum, Golgi apparatus and on the cell membrane. Hsp70 enhanced Kv1.5 current of transfected cells, which was abolished by pretreatment with brefeldin A or colchicine. Thus, Hsp70, but not other Hsps, stabilizes functional Kv1.5 protein.