Effects of emodin and vitamin C on growth performance, biochemical parameters and two HSP70s mRNA expression of Wuchang bream (Megalobrama amblycephala Yih) under high temperature stress

In order to study the effects of dietary emodin, high-dose vitamin C (Vc) and their combination on growth of Wuchang bream (Megalobrama amblycephala Y.) and its resistance to high temperature stress, 1200 healthy Wuchang bream with initial body weight of 133.44 ± 2.11 g were randomly divided into four groups: a control group fed with basal diet (containing 50.3 mg/kg Vc) and three treated groups fed with basal diets supplemented with 60 mg/kg emodin, 700 mg/kg Vc, and the combination of 60 mg/kg emodin + 700 mg/kg Vc, respectively. After feeding for 60 days, the growth performance of Wuchang bream was measured. Then 25 fish per tank were exposed to heat stress of 34 °C. The biochemical parameters of blood and liver, and expression levels of liver two HSP70s mRNA before and after heat stress were determined and the cumulative mortality of each group under heat stress was counted. The results showed that before stress, compared with the control, the weight gain (WG) and specific growth rate (SGR), serum total protein (TP), lysozyme (LSZ), and alkaline phosphatase (ALP) levels, liver superoxide dismutase (SOD) activity and expression level of HSP70 mRNA significantly increased in emodin and Vc groups while feed conversion rate (FCR), serum cortisol (COR), triglyceride (TG) and liver malondialdehyde (MDA) contents decreased (P < 0.05); liver catalase (CAT) activity also significantly increased in emodin group (P < 0.05). Although serum TP, LSZ, and liver HSP70 mRNA levels significantly increased and liver MDA level decreased in combination group (P < 0.05), no synergism was observed. After heat stress, compared with the control, the serum TP, LSZ, ALP levels, liver SOD, CAT activities, and expression levels of HSC70 and HSP70 mRNAs increased in emodin and Vc groups in varying degrees and serum COR, glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), TG and liver MDA levels decreased to some extent. Although these parameters had similar changing trend as above ones in combination group, it did not show any synergism either. Statistics showed that under heat stress, the cumulative mortalities of emodin and Vc groups, except at 6 h in emodin group, were significantly lower than that of the control (P < 0.05) while the difference between the combination and control groups was not significant (P > 0.05). Thus, the basal diet supplemented with 60 mg/kg emodin or 700 mg/kg Vc could promote the growth of Wuchang bream, reduce FCR, increase non-specific immunity of fish, antioxidant capacity, and two HSP70s mRNA expression levels, and enhance resistance to heat stress in fish. However, the combination of emodin and high-dose Vc showed no better effect.     

Structural organization of the spinach endoplasmic reticulum-luminal 70-kilodalton heat-shock cognate gene and expression of 70-kilodalton heat-shock genes during cold acclimation.

The 70-kD heat-shock proteins (HSP70s) are encoded by a multigene family in eukaryotes. In plants, the 70-kD heat-shock cognate (HSC70) proteins are located in organellar and cytosolic compartments of cells in most tissues. Previous work has indicated that HSC70 proteins of spinach (Spinacia oleracea) are actively synthesized during cold-acclimating conditions. We have isolated, sequenced, and characterized cDNA and genomic clones for the endoplasmic reticulum (ER) luminal HSC70 protein (immunoglobulin heavy chain-binding protein; BiP) of spinach. The spinach ER-luminal HSC70 is a constitutively expressed gene consisting of eight exons. Spinach BiP mRNA appears to be up-regulated during cold acclimation but is not expressed during water stress or heat shock. In contrast to the differential regulation of mRNA, the ER-luminal HSC70 protein levels remain constant in response to various environmental stresses. Two other members of the spinach 70-kD heat-shock (HS70) multigene family also show differential expression in response to a variety of environmental stresses. A constitutively expressed cytosolic HSC70 protein in spinach appears also to be up-regulated in response to both cold-acclimating and heat-shock treatments. Spinach also contains a cold-shock-induced HS70 gene that is not expressed during heat shock or water stress. Since HSP70s are considered to be involved with the chaperoning and folding of proteins, the data further support the concept that they may be important for maintaining cellular homeostasis and proper protein biogenesis during cold acclimation of spinach.     

Molecular identification and expression of heat shock cognate 70 (HSC70) in the Pacific white shrimp Litopenaeus vannamei

Heat shock protein 70s (HSP70s) are fundamental chaperone proteins that are indispensable to most living organisms. In order to investigate the function of HSP70 and heat shock response in shrimp, a heat shock cognate (HSC70) gene of the white shrimp (Litopenaeus vannamei), containing a 1959-bp open reading frame, was cloned and characterized. The amino acid sequence, 71.5 kDa of molecular weight, shares 80-99.6% homology with 12 diverse species' HSP70s and HSC70s. In fact, some segments of the eukaryotic HSC70 sequence, such as ATP/GTP-binding site, cytoplasmic HSP70 C-terminal sequence, and GGMP/GAP repeats, are also found in the putative shrimp HSC70. Moreover, multi-tissue RT-PCR was performed to assay the basal expressions of HSC70 in the heart, gill, hepatopancreas, stomach, gut, and muscle. The results demonstrate that the basal expressions of HSC70 in theses organs are similar to that of beta-actin. Furthermore, quantitative real-time experiments showed that HSC70 was up-regulated in hepatopancreas (4.6-fold), stomach (5.9-fold), gut (2.6-fold), and muscle (3.5-fold) but not in the heart (1.7-fold) and gill (1.6-fold) after 2 h of heat shock. Nevertheless, the HSC70 was found to be highly expressed in the heart and gill following 6 h of heat shock. This suggests that HSC70 in white shrimp possess both short-term and long-term responses to heat shock stress, indicating this HSC70 may be a heat-dependent HSC70 member. Finally, we constructed an expression vector to generate HSC70 in Escherichia coli BL21, which displayed immune cross-reactivity with mouse HSP70 antibody. In conclusion, the identification and expression of white shrimp HSC70 gene present useful data for studying the molecular mechanism of heat shock response and the effect of heat shock proteins in shrimps' cytoprotection.     

The Constitutive Heat Shock Protein-70 Is Required for Optimal Expression of Myelin Basic Protein During Differentiation of Oligodendrocytes

To further elucidate the role of the constitutive heat shock protein-70 (HSC70) as a chaperone for the synthesis of myelin basic protein (MBP), HSC70 content was decreased in oligodendrocyte precursor cells prior to MBP expression either by transfection with an antisense oligonucleotide specific for HSC70, or by exposure to low levels of quercetin, a bioflavonoid known to decrease synthesis of HSC70. As these cells underwent differentiation in vitro, antisense treatment decreased HSC70 levels to 66% of controls. At the same time, a sharp induction resulted in the stress-inducible heat shock protein-70 (HSP70). Levels of two other stress proteins increased as well, namely, the 25-kDa heat shock protein (HSP25) and the 78-kDa glucose regulated protein (GRP78). MBP synthesis proceeded over a normal time course, but at only 50% of control values. As HSC70 content returned to normal, MBP synthesis was also restored to normal levels. Quercetin reduced the expression of HSC70 to an even greater extent than transfection, and prevented the induction of HSP70. In contrast to antisense-treated cells, MBP synthesis was essentially blocked in quercetin-treated cells even though levels of HSP25 and GRP78 increased. Taken together, these observations (a) indicate that HSP70 partially compensates for decreased chaperoning of nascent MBP by HSC70 (HSC70 and HSP70 are closely related and perform similar functions); (b) preclude the involvement of HSP25 and GRP78 in MBP synthesis; and (c) emphasize the requirement of HSC70 for optimal synthesis of MBP.     

Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout

Heat shock proteins (HSPs) expression is commonly used as indicators of cellular stress in animals. However, very little is known about either the expression patterns of HSPs or their role in the stress-tolerance phenomenon in early life stages of fish. To this end, we examined the impact of food-deprivation (12 h), reduced oxygen levels (3.5 mg/L for 1 h) and heat shock (HS: + 5 °C for 1 h) on HSP70 and HSP90 protein expression in early life stages of the gilthead sea bream (Sparus aurata), a warm-water aquaculture species. Also, we investigated HSP70 and HSP90 response to food-deprivation (7 days) in early life stages of rainbow trout (Oncorhynchus mykiss), a cool-water aquaculture species, and the tolerance of this larvae to heat shock (either + 5 or + 10 °C for 1 h). Our results clearly demonstrate that food-deprivation enhances HSP70 and HSP90 protein expression in larvae of both species. In gilthead sea bream larvae, the stressors-induced HSP70 and HSP90 (only in the reduced oxygen group) protein expression returned to unstressed levels after 24 h recovery. In fed trout larvae, a + 5 °C heat shock did not elevate HSP70 and HSP90 expression, whereas 100% mortality was evident with a + 10 °C HS. However, food-deprived trout larvae, which had higher HSP70 and HSP90 protein content, survived HS and showed HS-dependent increases in HSP70, but not HSP90 expression. Overall, HSP70 and HSP90 protein expression in early life stages of fish have the potential to be used as markers of nutritional stress, while elevation of the tissue HSPs content may be used as a means to increase stress tolerance during larval rearing.     

Quantitative RT-PCR analysis and immunohistochemical localization of HSP70 in sea bass Dicentrarchus labrax exposed to transport stress

In aquaculture, fish are exposed to stressful conditions, which cause an increased synthesis of heat shock proteins (HSPs) at the cellular level. In this work we considered the expression of the constitutive and inducible forms of HSP70 as an indicator of stress caused by transport, during development of the sea bass (Dicentrarchus labrax), a teleost fish of high value for aquaculture. Qualitative RT-PCR analysis revealed expression of inducible HSP70 gene in larvae and fry (25, 40 and 80 days) as well as in adult tissues (liver, brain, muscle, gills, kidney, gonads, heart, spleen and skin) of both control and stressed animals. Expression of inducible HSP70 mRNA examined in different adult tissues by Real-Time PCR, was significantly higher in skin and skeletal muscle of stressed animals than in controls. Immunolocalization of inducible and constitutive forms of heat shock protein 70 (HSP70 and HSC70), reported here for the first time, demonstrated an ubiquitous distribution of HSC70 protein in several tissues of both stressed and control animals (at all stages), while inducible HSP70 protein was found only in skeletal muscle of stressed animals. In all stressed animals, regardless of their developmental stage, cortisol levels were higher than in control animals.     

Secretion of the heat shock proteins HSP70 and HSC70 by baby hamster kidney (BHK‐21) cells

The HSPs (heat‐shock proteins) of the 70‐kDa family, the constitutively expressed HSC70 (cognate 70‐kDa heat‐shock protein) and the stress‐inducible HSP70 (stress‐inducible 70‐kDa heat‐shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK‐21) cells released HSP70 and HSC70 in a serum‐free medium and that this process was the result of an active secretion of HSPs rather than the non‐specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non‐classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl‐β‐cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK‐21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK‐21 cells in a serum‐free medium through a non‐classical pathway in which lipid rafts play an important role.     

Reduction of HSP70 and HSC70 Heat Shock mRNA Induction by Pentobarbital After Transient Global Ischemia in Gerbil Brain

Abstract: The effect of pentobarbital on the induction of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient global ischemia in gerbil brains was investigated by in situ hybridization using cloned cDNA probes selective for each mRNA species. In sham control brains, HSP70 mRNA was scarcely present, whereas HSC70 mRNA was present in most cell populations. After a 5-min occlusion of bilateral common carotid arteries, HSP70 and HSC70 mRNAs were induced together in several cells and were especially dense in hippocampal dentate granule cells at 3 h, but the strong hybridization of the mRNAs continued only in hippocampal CA1 cells by 2 days. At 7 days after the ischemia, CA1 neuronal cell death was apparent, and the HSP70 mRNA disappeared and HSC70 mRNA content returned to the sham level, except for in the CA1 cells. Pretreatment with pentobarbital (40 mg/kg, i.p.) greatly reduced or inhibited the induction of HSP70 and HSC70 mRNAs at both early (3-h) and late (2-day) phases after ischemia. The drug also prevented CA1 cell death at 7 days along with the maintenance of expression of HSC70 mRNA at the sham control level. Hypothermic effects of pentobarbital were noted at 30 and 60 min after the reperfusion, whereas at 2 h there was no statistical significance between the control and drug-treated groups. The great reduction of HSP70 and HSC70 mRNA induction at both early and late phases after ischemia suggests that pentobarbital reduces intra- and/or postischemic stress and may protect CA1 cells from ischemic damage. These effects of the drug may be mainly due to its specific action rather than its hypothermic effects.     

黄颡鱼HSC70基因及其组织表达分析

热休克蛋白70（HSP70）与生物体的抗胁迫能力密切相关。本文采用RACE (Rapid amplification of cDNA ends) 技术,从黄颡鱼Pelteobagrus fulvidraco克隆到一种组成型热休克蛋白（HSC70）基因及其cDNA。该cDNA全长2245bp,包括5′非编码区82bp,3′非编码区225bp,开放阅读框(ORF) 1938bp,编码645个氨基酸组成的蛋白质。黄颡鱼HSC70基因含有8个内含子,与人、鼠、虹鳟和花斑溪鳉的HSC70基因内含子数目相同,位置相似。其中,最长内含子（873bp）位于5′端非编码区,其余内含子（长度在80－251bp之间不等）均在编码区以内。黄颡鱼HSC70基因编码的氨基酸序列与南方鲶的相似度最高,达96.13%,与欧洲银鲫和团头鲂的相似度分别为94.45%和94.14%。RT-PCR检测显示,正常情况下黄颡鱼HSC70在血细胞、心脏、肝、头肾、脾、鳃、肌肉和脑中均有表达,但表达量在鳃中最高,肌肉中最低;统计结果显示,热激后HSC70在血细胞、肝、头肾和脑中的表达量显著上升(p<0.05),而在其余组织中热激前后的表达差异不显著(p>0.05)。     

Characteristic changes of stress protein expression in streptozotocin-induced diabetic rats.

Stress proteins (heat shock proteins, HSP) play essential roles in folding, assembly and translocation of polypeptides and also in maintenance of the integrity of polypeptides as molecular chaperones. Since long-lasting hyperglycemia causes modification of cellular proteins, it is possible that expression of molecular chaperones may be altered during the course of diabetes. Here, we examined the cellular levels of stress proteins such as HSP105, HSP90 and HSC70/HSP70 in various tissues of streptozotocin-induced diabetic rats. In comparison to controls, the levels of HSC70 were markedly decreased in the liver but not in the brain, adrenal gland and pancreas of diabetic rats. The levels of HSP105 and HSP90 were not significantly changed in these tissues of diabetic rats. Furthermore, the induction of HSP70 as well as HSC70 by hyperthermia was significantly reduced in the liver and adrenal gland of diabetic rats. These results suggested that the expression and induction of HSC70/HSP70 may be altered during the course of diabetic disease and may result in impairment of the cytoprotective ability of diabetic rats.     

Molecular identification and expression of heat shock cognate 70 (<Emphasis Type="Italic">HSC</Emphasis>70) in the pacific white shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

Heat shock protein 70s (HSP70s) are fundamental chaperone proteins that are indispensable to most living organisms. In order to investigate the function of HSP70 and heat shock response in shrimp, a heat shock cognate (HSC70) gene of the white shrimp (Litopenaeus vannamei), containing a 1959-bp open reading frame, was cloned and characterized. The amino acid sequence, 71.5 kDa of molecular weight, shares 80–99.6% homology with 12 diverse species’ HSP70s and HSC70s. In fact, some segments of the eukaryotic HSC70 sequence, such as ATP/GTP-binding site, cytoplasmic HSP70 C-terminal sequence, and GGMP/GAP repeats, are also found in the putative shrimp HSC70. Moreover, multitissue RT-PCR was performed to assay the basal expressions of HSC70 in the heart, gill, hepatopancreas, stomach, gut, and muscle. The results demonstrate that the basal expressions of HSC70 in theses organs are similar to that of β-actin. Furthermore, quantitative real-time experiments showed that HSC70 was upregulated in hepatopancreas (4.6-fold), stomach (5.9-fold), gut (2.6-fold), and muscle (3.5-fold) but not in the heart (1.7-fold) and gill (1.6-fold) after 2 h of heat shock. Nevertheless, the HSC70 was found to be highly expressed in the heart and gill following 6 h of heat shock. This suggests that HSC70 in white shrimp possess both short-term and long-term responses to heat shock stress, indicating this HSC70 may be a heat-dependent HSC70 member. Finally, we constructed an expression vector to generate HSC70 in Escherichia coli BL21, which displayed immune cross-reactivity with mouse HSP70 antibody. In conclusion, the identification and expression of the white shrimp HSC70 gene present useful data for studying the molecular mechanism of heat shock response and the effect of heat shock proteins in shrimps’ cytoprotection. Published in Russian in Molekulyarnaya Biologiya, 2008, Vol. 42, No. 2, pp. 265–274. The text was submitted by the authors in English.     

The use of stress‐70 proteins in physiology: a re‐appraisal

There are few factors more important to the mechanisms of evolution than stress. The stress response has formed as a result of natural selection, improving the capacity of organisms to withstand situations that require action. The ubiquity of the cellular stress response suggests that effective mechanisms to counteract stress emerged early in the history of life, and their commonality proves how vital such mechanisms are to operative evolution. The cellular stress response (CSR) has been identified as a characteristic of cells in all three domains of life and consists of a core 44 proteins that are structurally highly conserved and that have been termed the ‘minimal stress proteome’ (MSP). Within the MSP, the most intensely researched proteins are a family of heat‐shock proteins known as HSP70. Superficially, correlations between the induction of stress and HSP70 differential expression support the use of HSP70 expression as a nonspecific biomarker of stress. However, we argue that too often authors have failed to question exactly what HSP70 differential expression signifies. Herein, we argue that HSP70 up‐regulation in response to stressors has been shown to be far more complex than the commonly accepted quasi‐linear relationship. In addition, in many instances, the uncertain identity and function of heat‐shock proteins and heat‐shock cognates has led to difficulties in interpretation of reports of inducible heat‐shock proteins and constitutive heat‐shock cognates. We caution against the broad application of HSP70 as a biomarker of stress in isolation and conclude that the application of HSP70 as a meaningful index of stress requires a higher degree of validation than the majority of research currently undertakes.     

Binding of Silurus asotus lectin to Gb3 on Raji cells causes disappearance of membrane-bound form of HSP70

Heat shock proteins (HSPs) are divided into stress-inducible and constitutive types. Generally, HSP70 (stress inducible) and HSC70 (constitutive) are representative of their types, respectively. From the results of immunocytochemical analysis, both HSP70 and HSC70 were constitutively expressed in globotriaosylceramide (Gb3)-expressing Raji cells as well as Gb3-negative K562 cells. Furthermore, the membrane-bound form of HSP70 was present on the surfaces of two cell lines as patch and cap-like structures, and was recovered in the cholesterol rich microdomains (CRM) prepared from them. On the other hand, HSP70 was partially co-localized with Gb3 on the surface of Raji cells. This result suggested that HSP70 was not associated with all of Gb3 molecules but with Gb3 specifically located in the particular environment. The effect of Silurus asotus lectin (SAL), which is one of the rhamnose-binding lectins and specifically binds to Gb3, on the disappearance of membrane-bound HSP70 was dependent on whether Gb3 was present or not. These results suggested that the disappearance of membrane-bound HSP70 was caused by SAL binding to Gb3, that the reduction of membrane-bound HSP70 might result in the decrease in cell volume observed, and that the mechanism of SAL-induced HSP70 expression may differ from that of heat shock in Raji cells.     

大菱鲆组成型热休克蛋白70的全长cDNA克隆及表达分析

根据感染哈维氏弧菌(Vibrio harveyi)的大菱鲆(Scophthalmus maximus)的差减cDNA文库中hsp70EST序列设计引物,用RACE方法首次克隆到长2188bp的大菱鲆HSC70(Heat-shock cognate protein 70)全长cDNA序列,包括1956bp的开放阅读框及5′和3′非翻译区。在预测的651个氨基酸序列中发现了Dnak特征性基序,胞质HSP70特征基序以及四肽简并重复序列。与其他真核生物HSP70家族成员进行同源性比较,发现大菱鲆HSC70与牙鲆(Paralichthys olivaceus)HSC71、虹鳟(Oncorhynchus mykiss)HSC71、人(Homo sapiens)HSC70、家鼠(Mus musculus)HSC70、烟草天蛾(Manduca sexta)HSC70的氨基酸相似性分别是97%,95%,94%,93%,86%,表现出较高的保守性。表达分析显示,hsc70mRNA在大菱鲆正常肝脏、鳃、肠、脾脏、头肾、肾等组织中以不同的水平存在,呈组成型表达;被哈维氏弧菌感染后,大菱鲆肝脏和脾脏组织hsc70mRNA表达水平分别在24h和12h出现上调(2.5-fold和1.6-fold);注射生理盐水组与未注射组之间差异不显著。       

Phenotypic plasticity of HSP70 and HSP70 gene expression in the Pacific oyster (Crassostrea gigas): implications for thermal limits and induction of thermal tolerance

Pacific oysters, Crassostrea gigas, living at a range of tidal heights, routinely encounter large seasonal fluctuations in temperature. We demonstrate that the thermal limits of oysters are relatively plastic, and that these limits are correlated with changes in the expression of one family of heat-shock proteins (HSP70). Oysters were cultured in the intertidal zone, at two tidal heights, and monitored for changes in expression of cognate (HSC) and inducible (HSP) heat-shock proteins during the progression from spring through winter. We found that the "control" levels (i.e., prior to laboratory heat shock) of HSC77 and HSC72 are positively correlated with increases in ambient temperature and were significantly higher in August than in January. The elevated level of HSCs during the summer was associated with moderate, 2-3 degrees C, increases in the upper thermal limits for survival. We measured concomitant increases in the threshold temperatures (T(on)) required for induction of HSP70. Total hsp70 mRNA expression reflected the seasonal changes in the expression of inducible but not cognate members of the HSP70 family of proteins. A potential cost of increased T(on) in the summer is that there was no extension of the upper thermal limits for survival (i.e., induction of thermotolerance) after sublethal heat shock at temperatures that were sufficient to induce thermotolerance during the winter months.