Identification of two hsp90 genes in carp

Two hsp90 cDNA isoforms (hsp90alpha and hsp90beta) were isolated from the common carp (Cyprinus carpio). Gene-specific probes and primers were selected and used in Northern blot hybridization and RT-PCR reactions to measure the basal hsp90 mRNA levels and to follow the inducer-specific expression of the hsp90 genes in different tissues during in vivo studies. The hsp90beta gene is largely constitutively expressed at a fairly high level in all the examined tissues (brain, liver and kidney) and is slightly inducible by an elevated temperature. Hsp90alpha mRNA is present in the brain, but is hardly detectable in the kidney and liver of unstressed animals. In the brain, this gene is greatly upregulated following thermal stress, whereas in the liver and kidney heat shock has only minor effects on its expression. Hsp90alpha, but not hsp90beta, responds to an elevated level of Cd in a dose-, time- and tissue-dependent manner.     

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.     

温度胁迫下棉花粉蚧热激蛋白基因的表达分析

【目的】为了研究热激蛋白 Hsp70, Hsp70-4和Hsp90在棉花粉蚧Phenacoccus solenopsis抵抗逆温中的作用。【方法】在测序棉花粉蚧转录组的基础上,分析了该虫热激蛋白Hsp70基因家族的2个序列［Pshsp70（GenBank登录号为KJ909505）和Pshsp70-4（GenBank登录号为KJ909506）］和Hsp90基因家族的1个序列,［Pshsp90(GenBank登录号为KJ909507)］,采用实时荧光定量 PCR（RT-qPCR）检测了在不同温度（18和32℃恒温, 37, 39, 41, 43和45℃热激1 h 后26℃恢复1 h)下棉花粉蚧不同发育阶段（2龄若虫、3龄若虫、雌成虫）3种热激蛋白基因的表达量。【结果】Pshsp70 cDNA序列包含1 923 bp的开放阅读框,编码641个氨基酸,理论分子量和等电点分别为70.9 kDa和5.65; Pshsp70-4 cDNA序列包含1 962 bp的开放阅读框,编码654个氨基酸,理论分子量和等电点分别为71.8 kDa和5.38;Pshsp90 cDNA序列包含2 172 bp的开放阅读框,编码724个氨基酸,理论分子量和等电点分别为83.5 kDa和4.93。Pshsp70 和Pshsp70-4均含有Hsp70基因家族高度保守的基序,Pshsp70编码的氨基酸序列与烟粉虱Bemisia tabaci和家蚕Bombyx mori等昆虫的Hsp70 的氨基酸序列一致性为 85%;Pshsp70-4编码的氨基酸序列与白蜡蚧Ericerus pela和点蜂缘蝽Riptortus pedestris等昆虫的Hsp70的氨基酸序列一致性高达95%;Pshsp90也含有Hsp90基因家族高度保守的基序,Pshsp90编码的氨基酸序列与赤拟谷盗Tribolium castaneum和东亚小花蝽Orius sauteri等昆虫的Hsp90 的氨基酸序列一致性为 87%。热激蛋白基因表达量分析结果表明,在18℃恒温条件下,粉蚧2龄若虫的3个PsHsps基因的mRNA相对表达量均比对照(26℃)低,在32℃恒温条件下,各龄期的Hsp70基因的相对表达量均显著高于对照。在37~45℃下热激1 h并在26℃下恢复1 h,棉花粉蚧3个龄期的3个热激蛋白PsHsps基因的相对表达量随温度的升高总体呈增加趋势,相关性分析表明,除Pshsp70-4在雌成虫中的表达量与热胁迫温度的相关系数为0.225外,各龄期中3个基因的表达量与温度的相关系数均大于0.6,显著相关;43℃和45℃胁迫下,各龄期的3个热激蛋白基因相对表达量均显著高于对照组（P<0.05）。【结论】棉花粉蚧热激蛋白基因的表达与温度呈正相关,在该虫应对高温中起着重要作用。     

Differential expression of Atlantic salmon thyrotropin β subunit mRNA and its cDNA sequence

To study the regulation of the thyroid system, an Atlantic salmon Salmo salar cDNA clone was isolated for thyroid stimulating hormone (TSH) β subunit gene. A cDNA (866 bp) was isolated from an adult Atlantic salmon pituitary cDNA library, this clone was sequenced and shown to be highly conserved when compared to other teleost β TSH subunit sequences. The cDNA was used as a probe for Northern blot analysis of total pituitary RNA from the different life cycle stages of Atlantic salmon. Northern blot analysis demonstrated that β TSH mRNA is expressed at all life cycle stages studied, including parr, smolt, immature fish at sea and sexually mature male fish. Densitometry of Northern blots showed that sexually mature male salmon had low levels of salmon β TSH mRNA compared to non-mature fish. Stunts, fish performing poorly in salt water, were shown to have elevated levels of β TSH mRNA when compared to healthy fish.     

Comparative expression analysis of two paralogous Hsp70s in rainbow trout cells exposed to heat stress

Heat-shock protein 70 (Hsp70) is the major stress-inducible protein in vertebrates and highly conserved throughout evolution. To accurately investigate the mRNA expression profiles of multiple Hsp70s in rainbow trout Oncorhynchus mykiss, we isolated full-length cDNA clones encoding Hsp70 from the fish and investigated their mRNA expression profiles during heat stress. Consequently, two Hsp70s, Hsp70a and Hsp70b, were identified and found to have 98.1% identity in their deduced amino acid sequences. Southern blot analysis indicated that the two Hsp70s are encoded by distinct genes in the genome. Northern blot analysis showed that each of Hsp70a and Hsp70b expressed two mRNA species having different sizes by heat stress in rainbow trout RTG-2 cells. The induction levels of total Hsp70b mRNAs were consistently higher than Hsp70a counterparts during heat stress, although the expression profiles of the two genes were similar to each other in temperature shift and time course experiments. Interestingly, an mRNA species with a larger molecular size was expressed only under severe heat stress not less than 28 degrees C irrespective of Hsp70a and Hsp70b. These results suggest that the comprehensive identification of duplicated genes is a prerequisite to examining the gene expression profiles for tetraploid species such as rainbow trout.     

灰飞虱热激蛋白Hsp90基因的克隆、序列分析与表达模式

【目的】灰飞虱Laodelphax striatellus (Fallén)是水稻上的重要害虫,它严重影响了水稻的产量和品质,特别由其传播的水稻条纹叶枯病毒对水稻的危害甚至更为严重。灰飞虱分布广,对环境有很强的适应性。本研究旨在探讨灰飞虱对温度胁迫适应的分子机制进行了初步的探讨。【方法】本研究采用RT-PCR与RACE技术克隆了热激蛋白Hsp90基因的全长,利用各种生物信息方法分析了该热激蛋白的特征,实时荧光定量PCR技术用于研究Hsp90基因在不同生长发育阶段和不同温度条件下表达变化规律。【结果】我们获得一条Hsp90 基因的cDNA全长序列,命名为LsHsp90（GenBank登录号为KF660250）。 LsHsp90全长为2 740 bp,编码729个氨基酸,等电点为5.0,分子量为83.7 kDa。氨基酸序列中含有 Hsp90蛋白家族的5个签名序列及胞质特征序列 MEEVD。结构预测表明LsHsp90具有Hsp90蛋白家族典型的结构特征。系统进化关系分析表明它与多种昆虫的Hsp90序列有较高的同源性。不同发育阶段的灰飞虱体内LsHsp90表达水平处于动态的变化过程中,并在4龄若虫期达到最大值,最低表达量在其雌成虫阶段。不同性别的灰飞虱成虫体内LsHsp90表达量有显著差异（P =0.008）。高温和低温胁迫都可以诱导灰飞虱体内LsHsp90的表达,最大的表达量分别在 40℃和-9℃。在40℃下处理不同时间后发现,LsHsp90的表达水平在处理后0.5 h时最高,但随着时间的延长,LsHsp90的表达水平逐渐降低。而在-4℃下,LsHsp90的表达水平在处理后1 h时达到最大值。【结论】灰飞虱体内的LsHsp90可以响应温度的诱导,它可能在灰飞虱温度胁迫适应过程中起到重要的作用。     

Presence of antibody against the inducible Hsp71 in patients with acute heat-induced illness

Antibodies against heat shock or stress proteins (Hsps) have been reported in a number of diseases in which they may be involved in the pathogenesis of the disease or may be of use for prognosis. Heat-induced diseases, such as heat cramps, heat exhaustion, or heat stroke, are frequent in hot working or living environments. There are still few investigations on the presence and possible significance of autoantibodies against Hsps in heat-induced illnesses. Using an immunoblotting technique with recombinant human Hsps, we analyzed the presence and titers of antibodies against Hsp60, Hsp71, and Hsp90alpha, and Hsp90beta in a group of 42 young male patients who presented with acute heat-induced illness during training. We also examined the presence of antibody against Hsp71 in a second group of 57 patients with acute heat-induced illness and measured the changes in titers of anti-Hsp71 antibodies in 9 patients hospitalized by emergency physicians. In the first group of young persons exercising in a hot environment, the occurrence of antibodies against Hsp71 and Hsp90alpha was significantly higher among individuals with symptoms of heat-induced illness (P < 0.05) than in the matched group of nonaffected exercising individuals. Moreover titers of antibody against Hsp71 were higher in individuals of the severe and mild heat-induced illness groups, the highest titer being found in the most severe cases. The results from the second group of 57 heat-affected patients exposed to extreme heat were similar. Again, patients with the more severe heat-induced symptoms showed a significantly higher incidence of antibodies to Hsp71 than controls and the titer of anti-Hsp71 was higher in the severely affected group. Finally, in a study of 9 patients, it was observed that the titer of anti-Hsp71 decreased during recovery from severe heat symptoms. These results suggest that measurement of antibodies to Hsps may be useful in assessing how individuals are responding to abnormal stress within their living and working environment and may be used as one biomarker to evaluate their susceptibility to heat-induced diseases.     

Identification of alpha-tubulin as an hsp105alpha-binding protein by the yeast two-hybrid system

Hsp105alpha is a mammalian stress protein that belongs to the HSP105/110 family. Hsp105alpha prevents stress-induced apoptosis in neuronal cells and binds to Hsp70/Hsc70 and suppresses the Hsp70 chaperone activity in vitro. In this study, to further elucidate the function of Hsp105alpha, we searched for Hsp105alpha-binding proteins by screening a mouse FM3A cell cDNA library with full-length Hsp105alpha using the yeast two-hybrid system and obtained alpha-tubulin as an Hsp105alpha-binding protein. Hsp105alpha bound directly to alpha-tubulin both in vitro and in vivo. Indirect immunofluorescence analysis with anti-Hsp105 and anti-alpha-tubulin antibodies indicated that Hsp105alpha was colocalized with microtubules. Furthermore, the disorganization of microtubules induced by heat shock was prevented in Hsp105alpha-overexpressing COS-7 cells. These findings suggested that Hsp105alpha associates with alpha-tubulin and microtubules in cells and plays a role in protection of microtubules under conditions of stress.     

Nucleotide sequence and growth hormone-regulated expression of salmon insulin-like growth factor I mRNA

Protein and cDNA sequence analysis have revealed that the insulin-like growth factor (IGF-I) has been highly conserved among several mammalian species. Using the combined techniques of polymerase chain reaction and molecular cloning, we have now obtained the cDNA sequence encoding preproIGF-I from a teleost species, Oncorhynchus kisutch (coho salmon). The 2020 nucleotide (nt) cloned cDNA sequence contains a 528 nt open reading frame encoding 176 amino acids in preproIGF-I and 175 nt and 1317 nt of flanking 5'- and 3'-untranslated regions, respectively. The deduced amino acid sequence of salmon IGF-I is highly conserved relative to its mammalian homologues and there are only 14 amino acid differences out of 70 between salmon and human IGF-I. Interestingly, the C-terminal E domain of salmon proIGF-I, which is presumed to be proteolytically cleaved during biosynthesis, also shows striking amino acid sequence homology with its mammalian counterpart, except for an internal 27 residue segment that is unique to salmon proIGF-I. Northern analysis revealed that salmon preproIGF-I mRNA consists predominantly of a single 3900 nt sized band although minor bands were also observed after prolonged autoradiographic exposure. The RNA analysis also revealed that the level of preproIGF-I mRNA is increased 6-fold in liver RNA isolated from salmon injected with bovine GH, as compared to untreated controls. These results demonstrate that the primary structure and regulated expression of IGF-I by GH have been conserved in teleosts.     

Intracellular localization of the 90 kDA heat shock protein (HSP90alpha) determined by expression of a EGFP-HSP90alpha-fusion protein in unstressed and heat stressed 3T3 cells

Heat shock protein 90 (Hsp90) is an abundant protein and essential for all eukaryotic cells. The expression of Hsp90 is further enhanced after exposure to stress factors, e.g. a heat shock. Many proteins interacting with Hsp90 as well as the various functions for Hsp90 have been described. In this study, an Hsp90alpha fusion protein along with the enhanced green fluorescence protein (EGFP) was expressed under the control of the human cytomegalovirus immediate early promoter. EGFP-Hsp90alpha was mainly localized in the cytoplasm, with only minor amounts inside the nuclei. No EGFP-Hsp90alpha could be detected inside the nucleoli. Following exposure to elevated temperatures, higher amounts of EGFP-Hsp90alpha are inside the nucleus, but not within the nucleoli. As the most remarkable finding under these conditions, an association of EGFP-Hsp90alpha with the nuclear membrane became visible.     

Cloning and characterization of the executioner caspases 3, 6, 7 and Hsp70 in hyperthermic Atlantic salmon (Salmo salar) embryos

Hyperthermia during embryogenesis has been reported to induce deformities in Atlantic salmon (Salmo salar). To examine the involvement of executioner caspases in hyperthermia-induced cell-death in a poikilotherm vertebrate species, five genes encoding caspase-3,-6, and -7 were cloned from Atlantic salmon, and the expression was studied in thermal stressed salmon embryos. The salmon genome contained two genetically distinct variants of both salmon caspase-3 and caspase-6 that is likely the result of two independent chromosome or genome duplications. Whereas only partial caspase-3A encoding sequences were isolated, the full-length caspase-3B cDNA encodes the inactive proenzyme of 279 amino acids (aa) consisting of an N-terminal prodomain and the large and the small subunit. The salmon caspase-6A and caspase-6B proenzymes include an additional linker region between the two subunits. The deduced salmon caspase-7 consists of only 245 aa and lacks the prodomain and part of the large subunit similar to the predicted caspase-7 of the puffer fish Tetraodon sp.. Increased apoptotic activity as evidenced by cleavage of nuclear DNA was demonstrated in salmon embryos incubated at 18-20 degrees C for 84 h after acclimatization at 8 degrees C. Hyperthermia-induced activation of the executioner caspases was indicated by the increased mRNA levels of caspase-3B, caspase-6A/B and caspase-7 after 54 h heat exposure as quantified by real-time RT-PCR. The 2-2.5 fold increase in the mRNA expression of the heat shock protein Hsp70 gene coincided with the peak mRNA values of the executioner caspases. Whole-mount in situ hybridization of the salmon embryo identified caspase-7 mRNA in the lens exclusively, while caspase-3B and caspase-6A/B were expressed in multiple tissues of exposed and control embryos. Interestingly, cardiac expression of caspase-6A/B was only identified in heat stressed embryos. Altogether, these results shed light on evolutionary aspects of the executioner caspases in vertebrates and their expression in salmon embryos exposed to hyperthermia. In particular, the heat sensitive caspase-6 expression in the embryonic heart is of interest since cardiac malformations are an emergent problem in salmon aquaculture.