• Journal of Life Science
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• v.28 no.6
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• pp.639-647
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• 2018

A new heat shock protein 70 was identified in red-spotted grouper (Epinephelus akaara) based on an expression analysis. The cDNA of red-spotted grouper Hsp70 (designated RgHsp70) was cloned by the rapid amplification of cDNA ends (RACE) techniques. The full-length of RgHsp70 cDNA was 2,152 bp, consisting of a 5'-terminal untranslated region (UTR) of 105 bp, a 3'-terminal UTR of 274 bp, and an open reading frame (ORF) of 1,773 bp that encode a polypeptide of 590 amino acids with a theoretical molecular weight of 64.9 kDa and an estimated isoelectric point of 5.2. Multiple alignment and phylogenetic analyses revealed that the RgHsp70 gene shares a high similarity with other Hsp70 fish genes. RgHsp70 contained all three classical Hsp70 family signatures. The results indicated the RgHsp70 is a member of the heat shock protein 70 family. RgHsp70 mRNA was predominately expressed in the liver, with reduced expression noted in the head-kidney tissues. The expression analysis of different water temperatures (21, 18, 15 and $12^{\circ}C$) for sampled livers revealed that expression gradually increased at $12^{\circ}C$ compared to $21^{\circ}C$. In this study, the effects of water temperature lowering on the physiological conditions were investigated, and the results revealed that novel RgHsp70 may be an important molecule involved in stress responses.

• Korean Journal of Plant Tissue Culture
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• v.28 no.2
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• pp.75-79
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• 2001

An experiment was carried out to introduce OsHSP17.9, a low molecular HSP gene isolated from rice plant to orchardgrass (Dactylis glomerata L.) using Agrobacterium. Mature seed-derived calli of orchardgrass were co-cultured with Agrobacterium tumefaciens EHA101 harboring the plasmid pIG-HSP17.9 for transformation. Calli selected by hygromycin were transferred to N$_{6}$ medium containing 1 mg/L NAA, 5 mg/L kinetin, 250 mg/L cefotaxime and 50 mg/L hygromycin and several hygromycin resistant plants were obtained. Stable incorporation of the introduced OsHSP17.9 to the genome of the hygromycin resistant plants was confirmed by PCR and Southern blot analysis. Transformation efficiency was variable between cultivars in which it was 16.5% in Potomac and 8.0% in Frontier. Constitutive expression of the transgene in the transformed orchardgrass tissues was identified by Northern blot analysis but transcript levels were different among individual plants.s.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.403-409
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• 2010

Saccharomyces cerevisiae Hsp30 is a plasma membrane heat shock protein that is induced by various environmental stress conditions. However, the functional role of Hsp30 during diverse environmental stressors is not presently known. To gain insight into its function during thermal stress, we have constructed and characterized a ${\Delta}hsp30$ strain during heat stress. $BY4741{\Delta}hsp30$ cells were found to be more sensitive compared with BY4741 cells, when exposed to a lethal heat stress at $50^{\circ}C$. When budding yeast is exposed to either heat shock or weak organic acid, it inhibits Pma1p activity. In this study, we measured the levels of Pma1p in mutant and Wt cells both during optimal temperature and heat shock temperature. We observed that $BY4741{\Delta}hsp30$ cells showed constitutive reduction of Pma1p. To gain further insights into the role of Hsp30 during heat stress, we compared the total protein profile by 2D gel electrophoresis followed by identification of differentially expressed spots by LC-MS. We observed that contrary to that expected from thermal-stress-induced changes in gene expression, the ${\Delta}hsp30$ mutant maintained elevated levels of Pdc1p, Trx1p, and Nbp35p and reduced levels of Atp2p and Sod1p during heat shock. In conclusion, Hsp30 is necessary during lethal heat stress, for the maintenance of Pma1p and a set of thermal stress response functions.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.2
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• pp.280-285
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• 2006

The current study was conducted to investigate the relationship between stress related gene and meat quality in pigs. A total number of 212 three-way cross bred (Landrace-$Yorkshire{\times}Duroc$) and 38 Duroc were sampled from the Korean pig industry to determine genotype frequency of porcine stress syndrome (PSS) and heat shock protein 70 kDa (HSP70) genes and their relationship with carcass traits and longissimus meat quality. Screen of HSP70 was performed by the single strand conformation polymorphism (SSCP) technique. Based on the analysis of restriction fragment length polymorphism (RFLP) in ryanodine receptor 1 (RYR1) gene, genetic disorder of PSS was related to a mutation at $18,168^{th}$ (C to T) of exon 17. There was no significant difference in ultimate meat pH and backfat thickness between HSP70 K1-AA type and -BB type in pure Duroc breed. In Landrace-$Yorkshire{\times}Duroc$ (L-$Y{\times}D$) cross bred pig, our results indicated that HSP70 derivate type in Duroc had a limited effect on backfat thickness, but L-$Y{\times}D$ type had a noticeable linkage with HSP70 K1-AA and K3-AB. This tendency was also observed in hot carcass weight where HSP70 K1-AA and K3-AB resulted in heavier weight with 86.3 kg compared to HSP70 K1-AB and K3-BB of 74.3 kg. Results imply that stress related HSP70 genotype has a potential association with backfat thickness and carcass weight.

• Genomics & Informatics
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• v.2 no.2
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• pp.67-74
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• 2004

Cells consistently face stressful conditions, which cause them to modulate a variety of intracellular processes and adapt to these environmental changes via regulation of gene expression. Hyperosmotic and oxidative stresses are significant stressors that induce cellular damage, and finally cell death. In this study, oligonucleotide microarrays were employed to investigate mRNA level changes in cells exposed to hyperosmotic or oxidative conditions. In addition, since heat shock protein 70 (HSP70) is one of the most inducible stress proteins and plays pivotal role to protect cells against stressful condition, we performed microarray analysis in HSP70-overexpressing cells to identify the genes expressed in a HSP70-dependent manner. Under hyperosmotic or oxidative stress conditions, a variety of genes showed altered expression. Down­regulation of protein phosphatase1 beta (PP1 beta) and sphingosine-1-phosphate phosphatase 1 (SPPase1) was detected in both stress conditions. Microarray analysis of HSP70-overexpressing cells demonstrated that diverse mRNA species depend on the level of cellular HSP70. Genes encoding Iysyl oxidase, thrombospondin 1, and procollagen displayed altered expression in all tested conditions. The results of this study will be useful to construct networks of stress response genes.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.1
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• pp.107-112
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• 2005

A high environmental temperature affects the economic performance of pigs. Heat shock protein 70 (HSP70) has been reported to participate importantly in thermotolerance. This study aims to produce transgenic pigs overexpressing porcine HSP70.2, the highly inducible one of HSP70 members, and to prove the cellular thermotolerance in the primary fibroblasts from the transgenics. A recombinant plasmid in which the sequence that encodes the porcine HSP70.2 gene is fused to green fluorescence protein (GFP) was constructed under the control of cytomegalovirus (CMV) enhancer and promoter. Two transgenic pigs were produced by microinjecting pCMV-HSP70-GFP DNA into the pronucleus of fertilized eggs. Immunoblot assay revealed the varied overexpression level (6.4% and 1.4%) of HSP70-GFP in transgenic pigs. After heating at $45^{\circ}C$ for 3 h, the survival rate (78.1%) of the primary fibroblast cells from the highly expressing transgenic pig exceeded that from the non-transgenic pig (62.9%). This result showed that primary fibroblasts overexpressing HSP70-GFP confer cell thermotolerance. We suggest that transgenic pigs overexpressing HSP70 might improve their thermotolerance in summer and therefore reduce the economic loss in animal production.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.33-45
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• 2011

Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bel-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MIT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG ($2{\mu}M$) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.

• International Journal of Industrial Entomology and Biomaterials
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• v.28 no.2
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• pp.71-84
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• 2014

The full-length heat shock protein 88 (HSP88) complementary DNA (cDNA) of Paecilomyces tenuipes Jocheon-1 was obtained by screening the Paecilomyces tenuipes (P. tenuipes) Jocheon-1 Uni-Zap cDNA library and performing 5' RACE polymerase chain reaction (PCR). The P. tenuipes Jocheon-1 HSP88 cDNA contained an open reading frame (ORF) of 2,139-basepair encoding 713 amino acid residues. The deduced amino acid sequence of the P. tenuipe s Jocheon-1 HSP88 cDNA showed 77% identity to Nectria haematococca HSP88 and 45-76% identity to other fungal homologous HSP88s. Phylogenetic analysis and BLAST program analysis confirmed that the deduced amino acid sequences of the P. tenuipes Jocheon-1 HSP88 gene belonged to the ascomycetes group within the fungal clade. The P. tenuipes Jocheon-1 HSP88 also contained the conserved ATPase domain at the N-terminal region. The cDNA encoding P. tenuipes Jocheon-1 HSP88 was expressed as an 88 kilodalton (kDa) polypeptide in baculovirus-infected insect Sf9 cells. Under higher temperature conditions for the growth of the entomopathogenic fungus, mRNA expression of P. tenuipes Jocheon-1 HSP88 was quantified by real time PCR (qPCR). The results showed that heat shock stress induced a higher level of mRNA expression compared to normal growth conditions.

• Journal of Life Science
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• v.21 no.5
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• pp.664-670
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• 2011

Oxidative stress results in sustained release of heat shock protein 90 (HSP90) from vascular smooth muscle cells (VSMCs). We investigated whether extracellular HSP90 predisposed VSMCs to pro-inflammatory phenotype. Exposure of human aortic smooth muscle cells to HSP90 not only significantly enhanced CXCL10 secretion but also increased CXCL10 transcription. HSP90-mediated CXCL10 secretion was attenuated by OxPAPC, a TLR-2/4 inhibitor, and curcumin, a TLR-4 dimerization inhibitor. Inhibitors of diphenyleneiodium chloride and the Akt pathway also attenuated CXCL10 secretion in response to HSP90. The gene delivery of I${\kappa}$B using recombinant adenoviruses and treatment with resveratrol, which inhibit NF-${\kappa}$B activity, significantly attenuated HSP90-induced CXCL10 secretion from VSMCs. We propose that extracellular HSP90 contributes to an inflammatory reaction in the stressed vasculature by inducing CXCL10 expression of VSMCs, and that TLR-4, Akt, and NF-${\kappa}$B play active roles in the process.

• Journal of The Korean Society of Grassland and Forage Science
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• v.20 no.2
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• pp.97-102
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• 2000

To increase thennotolerance of forage crops, transgenic rice plants as a model for transformation of monocots were generated. A cDNA encoding the chloroplast-localized small heat shock protein (small HSP) of rice, Oshsp21, was introduced into rice plants via Agrobacterium-mediated gene transfer system. Calli induced from scutella were co-cultivated with a A. tumefaciens strain EHAlOl canying a plasmid, pIGhsp21. A large number of transgenic plants were regenerated on a medium containing hygromycin. Integration of Oshsp2l gene was confirmed by PCR and Southern blot analyses with genomic DNA. Northern blot and immunoblot analyses revealed that the Oshsp21 gene was constitutively expressed and accumulated as mature protein in transgenic plants. Effects of constitutive expression of the OshspZl on thermotolerance were first probed with the chlorophyll fluorescence. Results indicate that inactivation of electron transport reactions in photosystem I1 (PSII), were mitigated by constitutive expression of the Oshsp21. These results suggest that the chloroplast small HSP plays an important role in protecting photosynthetic machinery during heat stress. (Key words : Thermotolerance, Rice, Transgenic, cDNA)