Expression of heat shock protein 60 in the tissues of transported piglets

In this study, we sought to determine the distribution and expression of heat shock protein 60 (Hsp60) in the tissues of transported piglets. A total of 24 Chinese Erhualian piglets with an average body weight of 20±1 kg were assessed under both 2-h transported and normal housing conditions. Results of enzymatic analysis showed that the serum creatine kinase and aspartate aminotransferase concentrations were significantly increased in the 2-h transported piglets. Acute cellular lesions characterized by granular and vacuolar degeneration of the parenchyma cells in the tested heart, liver, and kidney were also confirmed by histopathological test after 2 h transportation. These results indicate that transport stress induces tissue damage to heart, liver, and kidney. Hsp60-positive immunostaining was consistently detected in the cytoplasm of myocardial cells, hepatocytes, renal tubular epithelial cells, and epithelial cells of fundic gland. However, results of enzyme-linked immunosorbent assay indicated that Hsp60 expression was only significantly elevated in the stomach, with lower expression in the heart and a non-significant trend of increased liver and kidney expression of Hsp60. These results indicate that different tissues had different sensitivities to transport stress, possibly resulting in varying levels of cytoprotection by Hsp60 in the different tissues. The expression of Hsp60 following 2 h transportation coincided with deterioration of cardiac cytoprotection in the heart and protection in the stomach. However, the direct role of Hsp60 in cytoprotection of heart and stomach tissues needs further investigation.     

Genetic analysis of heat shock proteins in maize

A genetic analysis of heat shock protein (HSP) synthesis was performed in seedling leaf tissue of two maize inbred lines, their F₁ hybrid and F₂ progeny. Protein synthesis following a high temperature treatment was visualized by [³⁵S]-methionine in vivo labelling and two-dimensional gel electrophoresis. The parental lines' HSP synthesis patterns revealed both qualitative and quantitative polymorphisms implicative of differences in HSP structural genes and regulatory factors. The F₁ hybrid HSP profile indicated that synthesis of all parental HSPs conformed to dominant inheritance patterns, including complete dominance, over-dominance and co-dominance. Alleles for six low-molecularweight HSPs in F₂ progeny assorted according to typical 31 Mendelian ratios for dominant gene expression. There is evidence for unlinked gene loci of four different HSP gene pairs, but data for three other HSP gene pairs were inconclusive, perhaps reflecting linkage for one pair and complex regulatory factor interactions for the other two pairs of genes. These results clearly indicate the existence of genetic variability in HSP synthesis and emphasize the potential of partitioning their roles in thermal tolerance using genetic and molecular analyses.     

Thermal biology of horseshoe crab embryos and larvae: A role for heat shock proteins

Eggs of the American horseshoe crab, Limulus polyphemus L., develop on sandy estuarine beaches during the spring and summer, and are potentially vulnerable to thermal stress during the 3-4 weeks of development to the first instar (trilobite) larval stage. In many marine taxa, heat shock (stress) proteins (Hsp's) help individuals acclimate to stresses by restoring the proper folding of cellular proteins whose shape has been altered by temperature shock or other forms of environmental stress. We examined the survival of embryos and first instar (trilobite) larvae following heat shock, and compared the levels of Hsp70 in heat shocked and control animals. Animals acclimated to 13 or 22 °C had close to 100% survival when heat shocked for 3 h at 35 or 40 °C, but exposure to 45 °C for 3 h was lethal. To study the effect of heat shock on Hsp70 production under environmentally realistic conditions, animals were acclimated to either 13 or 22 °C, heat-shocked at 35 °C for 3 h, and soluble proteins were extracted following 0, 2, 4, or 6 h recovery at 22 °C. The relative amounts of Hsp70 in horseshoe crab embryos and larvae were examined using SDS-PAGE and Western blotting. Relative to controls animals held at a constant temperature, there was a slight elevation of Hsp70 only among heat shocked trilobite larvae in the 6 h recovery treatment. Hsp70 levels did not differ significantly between control and heat shocked embryos. Horseshoe crabs have adapted to living in a thermally stressful environment by maintaining a high baseline (constitutive) level of cellular stress proteins such as Hsp70, rather than by synthesizing inducible Hsp's when stressful temperatures are encountered. This may be an effective strategy given that the heat shocks encountered by intertidal embryos and larvae occur regularly as a function of diurnal and tidal temperature changes.     

Stress tolerance in a yeast sterol auxotroph: role of ergosterol, heat shock proteins and trehalose

The role of ergosterol in yeast stress tolerance, together with heat shock proteins (hsps) and trehalose, was examined in a sterol auxotrophic mutant of Saccharomyces cerevisiae. Ergosterol levels paralleled viability data, with cells containing higher levels of the sterol exhibiting greater tolerances to heat and ethanol. Although the mutant synthesised hsps and accumulated trehalose upon heat shock to the same levels as the wild-type cells, these parameters did not relate to stress tolerance. These results indicate that the role of ergosterol in stress tolerance is independent of hsps or trehalose.     

心血管系统热休克蛋白的研究进展

多种应激因素如热应激,缺血,血流动力学变化能引起细胞内代谢异常,细胞骨架紊乱等一系列的病理改变,同时细胞亦相应合成一系列分子量不同的热休克蛋白分子（HSPs)。研究表明,HSPs通过其分子伴侣功能,对细胞产生保护作用。近年来的研究发现,在心肌缺血,缺血预处理,心肌肥大和血管损伤等病理生理条件下,HSP70,HSP90,HSP47,HSP32,HSP27等热休克蛋白分子均参与心血管系统的保护作用。     

Heat shock proteins as an aid in the treatment and diagnosis of heat stroke

It is now well established that induction of heat shock protein 72 (HSP72) protects the cell or tissue against a second otherwise lethal exposure to heat, a phenomenon known as thermotolerance. Because of this protective role, HSP72 is potentially useful in the treatment of heat illnesses, which range from relatively benign disorders such as heat cramps to heat stroke, which can be life threatening. This review discusses various ways in which HSP72 might be used in the diagnosis and treatment of the heat illnesses. This includes methods to induce HSP72, analysis of HSP72 in the cells and tissues of heat stroke patients, and screening methods to detect individuals who may be heat intolerant.     

Proteomics analysis of two heat shock proteins in insects

Heat shock proteins (HSPs) are found in all living organisms, from bacteria to humans, are expressed under stress. In this study, characterization of two families of HSP including HSP60 and HSP70 protein was compared in different insect species from different orders. According to the conserved motifs analysis, none of the motifs were shared by all insects of two protein families but each family had their own common motifs. Functional and structural analyses were carried out on seven different insect species from each protein family as the representative samples. These analyses were performed via ExPASy database tools. The tertiary structure of Drosophila melanogater as the sample of each protein family were predicted by the Phyre2 and TM-score servers then their qualities were verified by SuperPose and PROCHECK. The tertiary structures were predicted through the “c4pj1E” model (PDB Accession Code: 4pj1) in HSP60 family and “c3d2fC” model (PDB Accession Code: 3d2f) in HSP70 family. The protein phylogenetic tree was constructed using the Neighbor-joining (NJ) method by Molecular Evolutionary Genetic Analysis (MEGA) 6.06. According to the results, there was a high identity of HSP60 and HSP70 families so that they should be derived from a common ancestor however they belonged to separate groups. In protein–protein interaction analysis by STRING 10.0, 10 common enriched pathways of biological process, molecular function and Kyoto Encyclopedia of Genes and Genomes (KEGG) were identified in D. melanogaster in both families. The obtained data provide a background for bioinformatic studies of the function and evolution of insects and other organisms.

Communicated by Ramaswamy H. Sarma     

Reversibility of heat shock in Chlamydia trachomatis

The heat shock effect on chlamydia development was studied. We report here that the reversibility of the heat shock response did not depend on the stage of chlamydial morphogenesis at which transfer to high temperature occurred, and the infectivity of the particles produced was not affected significantly, so long as the heat shock exposure was not prolonged. Exposure to heat shock for more than 9 h resulted in stagnation of the growth cycle, appearance of aberrant reticulate body particles and loss of infectivity. SDS-PAGE analysis of proteins synthesized under prolonged heat shock showed increased relative abundance of heat shock proteins in common with other procaryotic organisms.     

Low heat shock thresholds in wild Antarctic inter-tidal limpets (Nacella concinna)

Heat shock proteins (HSPs) are a family of genes classically used to measure levels of organism stress. We have previously identified two HSP70 genes (HSP70A and HSP70B) in sub-tidal populations of the Antarctic limpet (Nacella concinna). These genes are up-regulated in response to increased seawater temperatures of 15°C or more during acute heat shock experiments, temperatures that have very little basis when considering the current Antarctic ecology of these animals. Therefore, the question was posed as to whether these animals could express HSP70 genes when subjected to more complex environmental conditions, such as those that occur in the inter-tidal. Inter-tidal limpets were collected on three occasions in different weather conditions at South Cove, Rothera Point, over a complete tidal cycle, and the expression levels of the HSP70 genes were measured. Both genes showed relative up-regulation of gene expression over the period of the tidal cycle. The average foot temperature of these animals was 3.3°C, far below that of the acute heat shock experiments. These experiments demonstrate that the temperature and expression levels of HSP production in wild animals cannot be accurately extrapolated from experimentally induced treatments, especially when considering the complexity of stressors in the natural environment. However, experimental manipulation can provide molecular markers for identifying stress in Antarctic molluscs, provided it is accompanied by environmental validation, as demonstrated here. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cryopreservation disrupts lipid rafts and heat shock proteins in yellow catfish sperm

Lipid rafts and associated membrane proteins (flotillin, caveolin) play important roles in cell signaling and sperm fertilization while heat shock proteins (Hsp) ensure properly protein folding to fulfill their physiological functions. The markedly reduced fertility in thawed sperm after cryopreservation could result from disrupted membrane lipid rafts and these proteins. To explore the effect of sperm cryopreservation on lipid rafts and heat shock proteins, we compared lipid raft integrity, and the expression levels of lipid raft associated proteins (Flot-1, Flot-2, Cav-1) as well as heat shock proteins (Hsp90, Hsp70) in fresh and thawed sperm cryopreserved under different scenarios in yellow catfish. We found higher lipid raft integrity, higher protein expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 in fresh sperm samples than in thawed sperm samples, in thawed sperm samples cryopreserved with optimal cooling rate than those cryopreserved with sub-optimal cooling rate, and in thawed sperm samples cryopreserved with extenders supplemented with cholesterol than those supplemented with methyl-β-cyclodextrin (for cholesterol removal). Our findings indicate that lipid raft integrity, and expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 are clearly associated with sperm quality, and together they may play a cumulative role in reduced fertility associated with thawed sperm in aquatic species.     

Golgi fragmentation induced by heat shock or inhibition of heat shock proteins is mediated by non-muscle myosin IIA via its interaction with glycosyltransferases

The Golgi apparatus is a highly dynamic organelle which frequently undergoes morphological changes in certain normal physiological processes or in response to stress. The mechanisms are largely not known. We have found that heat shock of Panc1 cells expressing core 2 N-acetylglucosaminyltransferase-M (Panc1-C2GnT-M) induces Golgi disorganization by increasing non-muscle myosin IIA (NMIIA)–C2GnT-M complexes and polyubiquitination and proteasomal degradation of C2GnT-M. These effects are prevented by inhibition or knockdown of NMIIA. Also, the speed of Golgi fragmentation induced by heat shock is found to be positively correlated with the levels of C2GnT-M in the Golgi. The results are reproduced in LNCaP cells expressing high levels of two endogenous glycosyltransferases—core 2 N-acetylglucosaminyltransferase-L:1 and β-galactoside:α2-3 sialyltransferase 1. Further, during recovery after heat shock, Golgi reassembly as monitored by a Golgi matrix protein giantin precedes the return of C2GnT-M to the Golgi. The results are consistent with the roles of giantin as a building block of the Golgi architecture and a docking site for transport vesicles carrying glycosyltransferases. In addition, inhibition/depletion of HSP70 or HSP90 in Panc1-C2GnT-M cells also causes an increase of NMIIA–C2GnT-M complexes and NMIIA-mediated Golgi fragmentation but results in accumulation or degradation of C2GnT-M, respectively. These results can be explained by the known functions of these two HSP: participation of HSP90 in protein folding and HSP70 in protein folding and degradation. We conclude that NMIIA is the master regulator of Golgi fragmentation induced by heat shock or inhibition/depletion of HSP70/90.     

Antarctic marine molluscs do have an HSP70 heat shock response

The success of any organism depends not only on niche adaptation but also the ability to survive environmental perturbation from homeostasis, a situation generically described as stress. Although species-specific mechanisms to combat “stress” have been described, the production of heat shock proteins (HSPs), such as HSP70, is universally described across all taxa. Members of the HSP70 gene family comprising the constitutive (HSC70) and inducible (HSP70) members, plus GRP78 (glucose-regulated protein, 78 kDa), a related HSP70 family member, were cloned using degenerate polymerase chain reaction (PCR) from two evolutionary divergent Antarctic marine molluscs (Laternula elliptica and Nacella concinna), a bivalve and a gastropod, respectively. The expression of the HSP70 family members was surveyed via quantitative PCR after an acute 2-h heat shock experiment. Both species demonstrated significant up-regulation of HSP70 gene expression in response to increased temperatures. However, the temperature level at which these responses were induced varied with the species (+6–8°C for L. elliptica and +8–10°C for N. concinna) compared to their natural environmental temperature). L. elliptica also showed tissue-specific expression of the genes under study. Previous work on Antarctic fish has shown that they lack the classical heat shock response, with the inducible form of HSP70 being permanently expressed with an expression not further induced under higher temperature regimes. This study shows that this is not the case for other Antarctic animals, with the two molluscs showing an inducible heat shock response, at a level probably set during their temperate evolutionary past.     

Plasminogen and angiostatin interact with heat shock proteins

Previous studies from this laboratory have demonstrated that plasminogen and angiostatin bind to endothelial cell (EC) surface-associated actin via their kringles in a specific manner. Heat shock proteins (hsps) like hsp 27 are constitutively expressed by vascular ECs and regulate actin polymerization, cell growth, and migration. Since many hsps have also been found to be highly abundant on cell surfaces and there is evidence that bacterial surface hsps may interact with human plasminogen, the purpose of this study was to determine whether human plasminogen and angiostatin would interact with human hsps. ELISAs were developed in our laboratory to assess these interactions. It was observed that plasminogen bound to hsps 27, 60, and 70. In all cases, binding was inhibited (85–90%) by excess (50 mM) lysine indicating kringle involvement. Angiostatin predominantly bound to hsp 27 and to hsp 70 in a concentration- and kringle-dependent manner. As observed previously for actin, there was concentration-dependent inhibition of angiostatin’s interaction with hsp 27 by plasminogen. In addition, 30-fold molar excess actin inhibited (up to 50%), the interaction of plasminogen with all hsps. However, 30-fold molar excess actin could only inhibit the interaction of angiostatin with hsp 27 by 15–20%. Collectively, these data indicate that (i) while plasminogen interacts specifically with hsp 27, 60, and 70, angiostatin interacts predominantly with hsp 27 and to some extent with hsp 70; (ii) plasminogen only partially displaces angiostatin’s binding to hsp 27 and (iii) actin only partially displaces plasminogen/angiostatin binding to hsps. It is conceivable therefore that surface-associated hsps could mediate the binding of these ligands to cells like ECs.     

Expression of calcineurin,calpastatin and heat shock proteins during ischemia and reperfusion

ObjectiveCalcineurin (CaN) interacts with calpains (Calpn) and causes cellular damage eventually leading to cell death. Calpastatin (Calp) is a specific Calpn inhibitor, along with CaN stimulation has been implicated in reduced cell death and self-repair. Molecular chaperones, heat shock proteins (Hsp70 and Hsp90) acts as regulators in Calpn signaling. This study aims to elucidate the role of CaN, Calp and Hsps during induced ischemia and reperfusion in primary cardiomyocyte cultures (murine).Methods and resultsProtein expression was analyzed concurrently with viability using flow cytometry (FACS) in ischemia- and reperfusion-induced murine cardiomyocyte cultures. The expression of Hsp70 and Hsp90, both being molecular chaperones, increased during ischemia with a concurrent increase in death of cells expressing these proteins. The relative expression of Hsp70 and Hsp90 during ischemia with respect to CaN was enhanced in comparison to Calp. Reperfusion slightly decreased the number of cells expressing these chaperones. There was no increase in death of cells co-expressing Hsp70 and Hsp90 along with CaN and Calp. CaN expression peaked during ischemia and subsequent reperfusion reduced its expression and cell death. Calp expression increased both during ischemia and subsequent reperfusion but cell death decreased during reperfusion.ConclusionThe present study adds to the existing knowledge that Hsp70, Hsp90, CaN and Calp interact with each other and play significant role in cardio protection.     

PROGRESS STUDY: Progression of chronic kidney disease in children and heat shock proteins

Various molecular and cellular processes are involved in renal fibrosis, such as oxidative stress, inflammation, endothelial cell injury, and apoptosis. Heat shock proteins (HSPs) are implicated in the progression of chronic kidney disease (CKD). Our aim was to evaluate changes in urine and serum HSP levels over time and their relationships with the clinical parameters of CKD in children. In total, 117 children with CKD and 56 healthy children were examined. The CKD group was followed up prospectively for 24 months. Serum and urine HSP27, HSP40, HSP47, HSP60, HSP70, HSP72, and HSP90 levels and serum anti-HSP60 and anti-HSP70 levels were measured by ELISA at baseline, 12 months, and 24 months. The urine levels of all HSPs and the serum levels of HSP40, HSP47, HSP60, HSP70, anti-HSP60, and anti-HSP70 were higher at baseline in the CKD group than in the control group. Over the months, serum HSP47 and HSP60 levels steadily decreased, whereas HSP90 and anti-HSP60 levels steadily increased. Urine HSP levels were elevated in children with CKD; however, with the exception of HSP90, they decreased over time. In conclusion, our study demonstrates that CKD progression is a complicated process that involves HSPs, but they do not predict CKD progression. The protective role of HSPs against CKD may weaken over time, and HSP90 may have a detrimental effect on the disease course.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01239-9.     

Serum levels of heat shock protein 27 in patients with acute ischemic stroke

Expression of intracellular heat shock protein 27 (Hsp27) rises in the brain of animal models of cerebral ischemia and stroke. Hsp27 is also released into the circulation and the aim of the present study was to investigated if serum Hsp27 (sHsp27) levels are altered in patients with acute ischemic stroke. sHsp27 was measured in 15 patients with acute ischemic stroke and in 14 control subjects comparable for age, sex, and cardiovascular risk factors. In patients, measurements were performed at admission and 1, 2, and 30 days thereafter. At admission, mean sHsp27 values were threefold higher in patients than in controls. In patients, sHsp27 values dropped after 24 h, rose again at 48 h, and markedly declined at 30 days, indicating the presence of a temporal trend of sHsp27 values following acute ischemic stroke.