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.     

Immunocytochemical mapping of the novel echinoderm neuropeptide SALMFamide 1 (S1) in the starfish Asterias rubens

The recent isolation and characterization of the SALMFamide neuropeptides S1 and S2 from the starfish Asterias rubens has initiated a series of studies on their distribution. Specific antisera have been raised against S1 and used in light-microscopical immunocytochemistry. The results of this study reveal for the first time a possible hyponeural innervation of the visceral musculature of the gut and the widespread neuronal distribution of S1, (i) in axons and cell bodies of both ectoneural and hyponeral regions of the radial nerve cord and circumoral nerve ring, (ii) in the nerve ring and nerve plexus of the tube feet, (iii) in the apical muscle, (iv) in skin, and (v) extensively throughout the digestive system. These discoveries are of particular interest in terms of the possible functional roles for S1 in Asterias rubens.Part of this work has been previously reported as an abstract: (Moore et al.1990, 1991)     

Hypoxia modulates expression of the 70-kD heat shock protein and reduces<Emphasis Type="Italic">Leishmania</Emphasis> infection in macrophages

Hypoxia, a microenvironmental factor present in diseased tissues, has been recognized as a specific metabolic stimulus or a signal of cellular response. Experimental hypoxia has been reported to induce adaptation in macrophages such as differential migration, elevation of proinflammatory cytokines and glycolytic enzyme activities, and decreased phagocytosis of inert particles. In this study we demonstrate that although exposure to hypoxia (5% O₂, 5% CO₂, and balanced N₂) did not change macrophage viability, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cleavage and proliferation, it significantly reduced expression of the 70-kD heat shock protein (HSP70), which was restored to prehypoxia levels after reoxygenation. The influence of low oxygen tension on macrophage functional activity was also studied, i.e. the ability of these cells to maintain or resist infection by a microorganism. We demonstrate that macrophages from two different sources (a murine cell line and primary cells) exposed to hypoxia were efficiently infected withLeishmania amazonensis, but after 24 h showed a reduction in the percentage of infected cells and of the number of intracellular parasites per macrophage, indicating that hypoxia induced macrophages to kill the intracellular parasites. These results support the notion that hypoxia, a microenvironmental factor, can modulate macrophage protein expression and functional activity.     

HSP86 and HSP84 exhibit cellular specificity of expression and co-precipitate with an HSP70 family member in the murine testis

This study extends to the protein level our previous observations, which had established the stage and cellular specificity of expression of hsp86 and hsp84 in the murine testis in the absence of exogenous stress. Immunoblot analysis was used to demonstrate that HSP86 protein was present throughout testicular development and that its levels increased with the appearance of differentiating germ cells. HSP86 was most abundant in the germ cell population and was present at significantly lower levels in the somatic cells. By contrast, the HSP84 protein was detected in the somatic cells of the testis rather than in germ cells. The steady-state levels of HSP86 and HSP84 paralleled the pattern of the expression of their respective mRNAs, suggesting that regulation at the level of translation was not a major mechanism controlling hsp90 gene expression in testicular cells. Immunoprecipitation analysis revealed that a 70-kDa protein coprecipitated with the HSP86/HSP84 proteins in testicular homogenates. This protein was identified as an HSP70 family member by immunoblot analysis, suggesting that HSP70 and HSP90 family members interact in testicular cells. © 1993Wiley-Liss, Inc.     

Seasonal variation in expression pattern of genes under HSP70

Heat shock protein 70 (HSP70) is one of the most abundant and best characterized heat shock protein family that consists of highly conserved stress proteins, expressed in response to stress, and plays crucial roles in environmental stress tolerance and adaptation. The present study was conducted to identify major types of genes under the HSP70 family and to quantify their expression pattern in heat- and cold-adapted Indian goats (Capra hircus) with respect to different seasons. Five HSP70 gene homologues to HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2 were identified by gene-specific primers. The cDNA sequences showed high similarity to other mammals, and proteins have an estimated molecular weight of around 70 kDa. The expression of HSP70 genes was observed during summer and winter. During summer, the higher expression of HSPA8, HSPA6, and HSPA1A was observed, whereas the expression levels of HSPA1L and HSPA2 were found to be lower. It was also observed that the expression of HSPA1A and HSPA8 was higher during winter in both heat- and cold-adapted goats but downregulates in case of other HSPs. Therefore, both heat and cold stress induced the overexpression of HSP70 genes. An interesting finding that emerged from the study is the higher expression of HSP70 genes in cold-adapted goats during summer and in heat-adapted goats during winter. Altogether, the results indicate that the expression pattern of HSP70 genes is species- and breed-specific, most likely due to variations in thermal tolerance and adaptation to different climatic conditions.     

Cellular distribution of Hsp70 expression in rat skeletal muscles. Effects of moderate exercise training and chronic hypoxia

Rat hindlimb muscles constitutively express the inducible heat shock protein 72 (Hsp70), apparently in proportion to the slow myosin content. Since it remains controversial whether chronic Hsp70 expression reflects the overimposed stress, we investigated Hsp70 cellular distribution in fast muscles of the posterior rat hindlimb after (1) mild exercise training (up to 30 m/min treadmill run for 1 h/day), which induces a remodeling in fast fiber composition, or (2) prolonged exposure to normobaric hypoxia (10%O₂), which does not affect fiber-type composition. Both conditions increased significantly protein Hsp70 levels in the skeletal muscle. Immunohistochemistry showed the labeling for Hsp70 in subsets of both slow/type 1 and fast/type 2A myofibers of control, sedentary, and normoxic rats. Endurance training increased about threefold the percentage of Hsp70-positive myofibers (P<0.001), and changed the distribution of Hsp70 immunoreactivity, which involved a larger subset of both type 2A and intermediate type 2A/2X myofibers (P<0.001) and vascular smooth muscle cells. Hypoxia induced Hsp70 immunoreactivity in smooth muscle cells of veins and did not increase the percentage of Hsp70-positive myofibers; however, sustained exposure to hypoxia affected the distribution of Hsp70 immunoreactivity, which appeared detectable in a very small subset of type 2A fibers, whereas it concentrated in type 1 myofibers (P<0.05) together with the labeling for heme-oxygenase isoform 1, a marker of oxidative stress. Therefore, the chronic induction of Hsp70 expression in rat skeletal muscles is not obligatory related to the slow fiber phenotype but reveals the occurrence of a stress response.     

Localisation of gamma aminobutyric acid (GABA)-like immunoreactivity in the echinoderm Asterias rubens

Gamma amino butyric acid (GABA) is believed to be the principal inhibitory neurotransmitter in the mammalian central nervous system, a function that has been extended to a number of invertebrate systems. We have used a specific antiserum raised against GABA to demonstrate GABA-like immunoreactivity in the radial nerve cord (RNC), tube feet and the digestive system of the asteroid Asterias rubens. In the RNC, immunoreactivity was restricted to ectoneural fibres and cell bodies while in the tube feet fibres were revealed in the basal nerve ring and longitudinal nerve. In the gut, extensive labelling was apparent in the basi-epithelial plexus as well as in mucosal perikarya.     

Comparative analysis of differential gene expression of HSP40 and HSP70 family isoforms during heat stress and HIV-1 infection in T-cells

Heat shock proteins (HSPs) are a family of cellular proteins involved in a variety of biological functions including chaperone activity. HSPs are classified based on their molecular weight and each family has several isoforms in eukaryotes. HSP40 is the most diverse family acting as a co-chaperone for the highly conserved HSP70 family. Some of the isoforms are reported to be induced during heat stress. Few studies have also highlighted the diverse role of some isoforms in different stress conditions including viral infections. But till date, no study has comprehensively examined the expression profile of different HSP40 and 70 isoforms in either heat stress or HIV-1 infection, a virus that is responsible for the pandemic of AIDS. In the present study, we have compared the mRNA expression profile of HSP40 and HSP70 isoforms during heat stress and HIV-1 infection in a T-cell line and also validated the HIV-1 stress results in peripheral blood mononuclear cells. In case of HSP70, we observed that three isoforms (HSPA1A, HSPA1B, and HSPA6) are highly upregulated during heat stress, but these isoforms were found to be downregulated during the peak of HIV-1 infection. While in case of HSP40, we found that only DNAJA4, DNAJB1, and DNAJB4 showed significant upregulation during heat stress, whereas in HIV-1 infection, majority of the isoforms were induced significantly. Stress-dependent differential expression observed here indicates that different HSP40 and HSP70 isoforms may have specific roles during HIV-1 infection and thus could be important for future studies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-020-01185-y.     

热休克反应中小鼠心脏HSP70表达的免疫组织化学研究

用免疫组织化学方法研究了小鼠心脏在不同温度（40、41、44、46℃）热休克处理后,各恢复期（2、4、8、12、24小时）HSP70的表达。结果表明,（1）44、46℃热处理能诱导心肌细胞合成HSP70,以46℃为多（P＜0．01）,且于恢复期4－8小时为合成高峰（P＜0．01）。（2）阳性免疫反应定位于心肌细胞质中,核呈阴性反应。提示了心脏有较强的耐热能力。     

Litopenaeus vannamei oxygen consumption and HSP gene expression at cyclic conditions of hyperthermia and hypoxia

Although Litopenaeus vannamei is a widely studied species, the information on how the organisms respond to natural daily variations of environmental conditions such as temperature and dissolved oxygen, and how such conditions alter the physiological responses, is scarce. In the present work, the strategies used by shrimps to cope with temperature and dissolved oxygen fluctuations during 24 days were investigated through the evaluation of oxygen consumption and heat shock proteins (HSP) gene expression. During daily fluctuations, no change in oxygen consumption in the short-term, but a significant increase in the long-term during hyperthermia conditions was registered, whereas a significant decrease during hypoxia was observed during all the bioassay. On the other hand, HSP70 and HSP90 gene expression increased in gills under thermal stress but was down-regulated under hypoxia, in both the short- and the long-term. This study highlights that to counteract environmental variations of temperature and dissolved oxygen, the shrimps use molecular compensatory mechanisms (HSP gene expression) that are different to those used under constant hypoxic conditions, suggesting that hypoxia can compromise physiological cytoprotection.     

Regulation of HSP70 and HSP28 gene expression: absence of compensatory interactions

We have previously reported the lack of HSP28 gene expression during acute and chronic thermotolerance development in L929 cells (J Cell Physiol 152: 118–125, 1992; Cancer Res 52: 5787, 1992). In contrast to HSP28, an extremely high level of inducible HSP70 synthesis was observed. These results led us to investigate the possibility of compensatory interactions between HSP70 and HSP28. To test the hypothesis, L929 cells were transfected with the human HSP28 gene contained in plasmid pCMV27. Data from Western blot and two-dimensional gel electrophoresis of [³H] leucine and [³²P] orthophosphate-labeled proteins showed the synthesis and phosphorylation of HSP28 in transfected cells after heating at 45°C for 10 min. However, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, was not decreased after heat shock. These results suggest an independent regulation of HSP28 and HSP70 gene expression.     

Cytoplasmic HSP70 homologues of pea: differential expression in vegetative and embryonic organs

Eukaryotes express several cytoplasmic HSP70 genes, and their encoded proteins participate in diverse cellular processes. Three cDNAs encoding highly expressed cytoplasmic HSP70 homologues from Pisum sativum were cloned and characterized. They were designated PsHSP71.2, PsHSC71.0, and PsHSP70b. These HSP70 genes have different expression profiles in leaves: PsHSP71.2 is observed only in response to heat stress, PsHSC71.0 is present constitutively, and PsHSP70b is weakly constitutively expressed, but induced strongly in response to heat stress. In addition to being heat induced, the PsHSP71.2 mRNA is also expressed in zygotic, but not maternal organs of developing pea seeds, while PsHSC71.0 and PsHSP70b mRNAs are present in maternal and zygotic organs throughout seed development. Immunoblot analysis of parallel protein samples detects a 70 kDa polypeptide in all samples, and a 72 kDa polypeptide that corresponds to the PsHSP71.2 gene product is observed in cotyledons beginning at mid-maturation and in axes beginning between late maturation and desiccation. This polypeptide is not detected in the seed coat. The 72 kDa polypeptide remains abundant in both cotyledons and axes through germination, but declines substantially between 48 and 72 h after the onset of imbibition. Differential control of HSP70 expression during heat stress, seed maturation, and germination is consistent with the hypothesis that there are functional distinctions between cytoplasmic HSP70s.     

Nickel, lead, and cadmium induce differential cellular responses in sea urchin embryos by activating the synthesis of different HSP70s

Treatment with heavy metals, such as nickel, lead or cadmium, elicits different cellular stress responses according to the metal used and the length of treatment. In Paracentrotus lividus embryos the inducible forms of HSP70 (HSP70/72) are different in molecular mass from the constitutively expressed HSP75, and they can be used as markers of cellular stress. Even a short treatment with each metal induces the synthesis of HSP70/72 which remain stable for at least 20h and differ little in their isoelectric points. Continuous treatment from fertilization with nickel or lead produces late irregular pluteus embryos, with peak HSP70/72 synthesis at blastula followed by the arrest of synthesis by pluteus. On the contrary, the same treatment with cadmium induces continuous HSP70/72 synthesis and produces irregular gastrula embryos which then degenerate. Moreover, a long treatment induces over control embryos a slight increase in the amount of constitutive HSP75 during development while lead treatment depresses constitutive HSP75 at early stages and doubles its quantity at late stages.     

Modulation of ASK1 expression during overexpression of Trx and HSP70 in stressed fish liver mitochondria

Mitochondrial heat shock protein 70 (mtHSP70) is found to play a primary role in cellular defense against physiological stress like exposure to environmental contaminants and helpful in the maintenance of cellular homeostasis by promoting the cell survival. In the present investigation, the environmental-stress-induced increase in mtHSP70 levels along with the quantification of apoptosis signal regulating kinase 1 (ASK1) and thioredoxin (Trx) were measured in the liver mitochondria of grey mullets (Mugil cephalus) collected from the polluted Ennore estuary and the unpolluted Kovalam estuary for a period of 2 years. The results showed elevated lipid peroxide (LPO) and decreased total antioxidant capacity along with the decrease in mitochondrial viability percentage. Mitochondrial HSP70, ASK1, and Trx levels were increased under this stress condition. A 42% increase in LPO levels and 18% decrease in mitochondrial survivality were observed in the polluted-site fish liver mitochondria when compared to the results of unpolluted estuary. We also report that, under observed oxidative stress condition in Ennore fish samples, the ASK1 levels are only moderately elevated (13% increase). This may be due to mitochondrial-HSP70-induced adaptive tolerance signaling for the activation of Trx (22% increase) which suppresses the ASK1 expression thereby promoting the cell survival that leads to the maintenance of the cellular homeostasis.     

奶牛HSP70基因表达及其连锁微卫星标记与耐热性状的相关性

为了研究奶牛HSP70基因的mRNA表达规律和其紧密连锁的3个微卫星标记与耐热性状的相关性,文中在日平均温湿度指数(THI)为86.2(高温期)、70.9(临界高温期)和56.8(适温期)时分别采集10头处于同一泌乳阶段的同龄健康奶牛的尾静脉血,采用实时荧光定量RT-PCR技术分析不同条件下奶牛HSP70基因在外周血淋巴细胞的mRNA表达水平。结果表明:随THI升高,HSP70mRNA表达量呈上升趋势,高温期奶牛的外周血淋巴细胞HSP70mRNA表达丰度极显著高于临界高温期和适温期(P0.01),且与奶牛的耐热指标具有一定的遗传相关,表明HSP70基因可以作为奶牛热应激反应的候选基因。选取23号染色体上与HSP70紧密连锁的3个微卫星标记BMS468、BM1258和BM1815,用非变性聚丙烯酰胺凝胶电泳分析其在160头荷斯坦奶牛的遗传变异情况,利用最小二乘法拟合线性模型初步探索了这3个微卫星座位与奶牛耐热指标的关系。相关分析表明:耐热系数、红细胞钾含量及高温期日产奶量下降率的最有利基因型在BMS468基因座和BM1815基因座上,分别为134bp/128bp(P0.05)和186bp/148bp(P0.05);BM1258基因座上,最有利基因型101bp/99bp的高温期日产奶量下降率最低(P0.05)。     

饥饿对小鼠脑组织中转氨酶、肌酸激酶、乳酸脱氢酶和热休克蛋白70的影响

饥饿处理小鼠1～3天,分别检测脑组织中转氨酶、肌酸激酶、乳酸脱氧酶和HSPT0的变化.结果表明,谷丙转氨酶在饥饿1～3天有逐渐增高的趋势;谷草转氨酶在饥饿1～2天明显升高,但饥饿3天后迅速下降;在正常脑组织中,肌酸激酶的活性较强,饥饿1天后迅速下降,2～3天维持在较低水平;乳酸脱氢酶和HSP70受饥饿影响明显升高.