Heat shock proteins (HSPs) widely exist in all organisms, the structures of which are usually extraordinarily conservative. They are also well-known stress proteins that are involved in response to physical, chemical and biological stresses. HSP70 is an important member of the HSPs family. In order to study the roles of amphibians HSP70 during infection, the cDNA sequence of Rana amurensis hsp70 family genes were cloned by homologous cloning method. The sequence characteristics, three-dimensional structure and genetic relationship of Ra-hsp70s were analyzed by bioinformatics methods. The expression profiles under bacterial infection were also analyzed by real-time quantitative PCR (qRT-PCR). Expression and localization of HSP70 protein were tested by immunohistochemical techniques. The results showed that three conservative tag sequences of HSP70 family, HSPA5, HSPA8 and HSPA13, were found in HSP70. Phylogenetic tree analysis indicated four members are distributed in four different branches, and members with the same subcellular localization motif are distributed in the same branch. The relative expression levels of the mRNA of four members were all significantly upregulated (P < 0.01) upon infection, but the time for up-regulating the expression levels were diverse in different tissues. The immunohistochemical analysis showed that HSP70 was expressed to different degrees in the cytoplasm of liver, kidney, skin and stomach tissue. The four members of Ra-hsp70 family have ability to respond bacterial infection to varying degrees. Therefore, it was proposed that they are involved in biological processes against pathogen and play different biological functions. The study provides a theoretical basis for functional studies of HSP70 gene in amphibians.
Heat-Shock Proteins/genetics* ; Phylogeny ; Amino Acid Sequence ; HSP70 Heat-Shock Proteins/metabolism* ; Stress, Physiological

Heat stress can stimulate an increase in body temperature, which is correlated with increased expression of heat shock protein 70 (HSP70) and tumor necrosis factor α (TNFα). The exact mechanism underlying the HSP70 and TNFα induction is unclear. Berberine (BBR) can significantly inhibit the temperature rise caused by heat stress, but the mechanism responsible for the BBR effect on HSP70 and TNFα signaling has not been investigated. The aim of the present study was to explore the relationship between the expression of HSP70 and TNFα and the effects of BBR under heat conditions, using in vivo and in vitro models. The expression levels of HSP70 and TNFα were determined using RT-PCR and Western blotting analyses. The results showed that the levels of HSP70 and TNFα were up-regulated under heat conditions (40 °C). HSP70 acted as a chaperone to maintain TNFα homeostasis with rising the temperature, but knockdown of HSP70 could not down-regulate the level of TNFα. Furthermore, TNFα could not influence the expression of HSP70 under normal and heat conditions. BBR targeted both HSP70 and TNFα by suppressing their gene transcription, thereby decreasing body temperature under heat conditions. In conclusion, BBR has a potential to be developed as a therapeutic strategy for suppressing the thermal effects in hot environments.
Animals ; Berberine ; pharmacology ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; Heat Stress Disorders ; drug therapy ; genetics ; metabolism ; Hot Temperature ; Humans ; Male ; Mice ; Mice, Inbred ICR ; TATA Box ; drug effects ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVEThe objective was to observe damage of hippocampus in rats after exposure to infrasound, and to assess HSP70 expression in hippocampus.

METHODSSD rats in the experimental group were exposed to 140 dB (8 Hz) infrasound for 2 h per day for 3 days. The morphology of the hippocampus was examined by transmission electronic microscopic (TEM). Cell apoptosis was observed by TUNEL staining at 0 h, 24 h, 48 h, and 2 w after exposure. HSP70 expression was detected by immunohistochemistry (IHC) and Western blotting (WB).

RESULTSTEM showed that hippocampus was significantly damaged by exposure, and exhibited recovery 1 week after exposure. The TUNEL data showed that neuronal apoptosis after exposure was significantly higher than in the control rats at 24 h and 48 h, and the apoptotic cells decreased one week after exposure. IHC and WB showed HSP70 expression was significantly higher in the exposed rats, peaked at 24 h.

CONCLUSIONExposure to 140 dB (8 Hz) infrasound for 2 h per day for 3 days appeared to induce damage to the hippocampus of rats, based on changes in ultrastructure and increased cell apoptosis. However, recovery from the damage occurred overtime. HSP70 expression also increased after the exposure and decreased by 48.

Animals ; Apoptosis ; Blotting, Western ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; Hippocampus ; radiation effects ; Immunohistochemistry ; In Situ Nick-End Labeling ; Male ; Rats ; Rats, Sprague-Dawley ; Sound ; adverse effects

OBJECTIVETo investigate the effect of heat shock protein 70 (HSP70) on pulmonary arterial pressure and pulmonary vascular remodeling in neonatal rats with hypoxic pulmonary hypertension (HPH).

METHODSA total of 128 Wistar neonatal rats were randomly divided into HPH model and blank control groups. According to the transfection solution, the HPH model group was further divided into normal saline group, empty virus group (viral vectors marked with a green fluorescent signal and not carrying the target gene), and virus+HSP70 group (viral vectors marked with a green fluorescent signal and carrying the target gene). The HPH model was established by inhalation of nitrogen-oxygen mixture (1.5 L/minutes and 8% oxygen). Pulmonary arterial pressure (mPAP) and the indicators of pulmonary vascular remodeling (MT% and MA%) were measured on days 3, 7, 10, and 14 of hypoxia.

RESULTSOn days 3, 7, and 10 of hypoxia, the normal saline and empty virus groups had significantly enhanced expression of HSP70 compared with the blank control group (P<0.01), and the virus+HSP70 group had significantly higher expression of HSP70 than the blank control, normal saline, and empty virus groups (P<0.01). On day 14 of hypoxia, the expression of HSP70 showed no significant difference between these groups (P>0.05). On days 3, 7, and 10 of hypoxia, the normal saline and empty virus groups showed continuous increases in mPAP compared with the blank control group (P<0.05). There was no significant difference in mPAP between the virus+HSP70 and blank control groups (P>0.05). On day 14 of hypoxia, there was no significant difference in mPAP among three subgroups of the HPH model group (P>0.05), but the mPAP in the three subgroups was significantly higher than in the blank control group (P<0.05). After 7 days of hypoxia, the normal saline and empty virus groups showed significantly higher MT% and MA% than the blank control group (P<0.05), but the two indicators showed no significant differences between the virus+HSP70 and the blank control groups (P>0.05). On day 14 of hypoxia, there were no significant differences in MT% and MA% among three subgroups of the HPH model group (P>0.05), but the MT% and MA% in the three subgroups were higher than in the blank control group (P<0.05).

CONCLUSIONSHSP70 may reduce pulmonary arterial pressure and pulmonary vascular remodeling in neonatal rats with HPH.

Animals ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Hypertension, Pulmonary ; cerebrospinal fluid ; metabolism ; physiopathology ; Hypoxia ; genetics ; metabolism ; physiopathology ; Oxygen ; metabolism ; Pulmonary Artery ; metabolism ; Rats ; Rats, Wistar ; Vascular Remodeling

PURPOSE: There are currently no consistently effective treatments for the excessive collagen produced by keloid fibroblasts. Previously, we reported that heat shock protein 70 (Hsp70) is up-regulated in keloid fibroblasts and keloid tissue. We, therefore, investigated whether Hsp70 is related to excessive collagen production in keloid fibroblasts. MATERIALS AND METHODS: We inhibited Hsp70 in keloid fibroblasts by RNA interference and examined the resulting collagen expression. Thus, we selected small interfering RNAs (siRNAs) specific for human Hsp70, transfected them into keloid fibroblasts, and evaluated the resulting phenotypes and protein production using real-time polymerase chain reaction (PCR), Western blot, and a collagen assay. RESULTS: The siRNAs dramatically suppressed Hsp70 mRNA expression, resulting in a decrease in collagen production in the keloid fibroblasts compared with controls. The siRNAs did not influence the viability of the keloid fibroblasts. CONCLUSION: Hsp70 overexpression likely plays an important role in the excessive collagen production by keloid fibroblasts. RNA interference has therapeutic potential for the treatment of keloids.
Adolescent ; Adult ; Blotting, Western ; Collagen/*drug effects/metabolism ; Female ; Fibroblasts/metabolism ; Gene Expression Regulation ; HSP70 Heat-Shock Proteins/genetics/metabolism/*pharmacology ; Humans ; Keloid/*drug therapy/genetics/metabolism ; Male ; RNA, Messenger/*genetics ; RNA, Small Interfering/*genetics ; Real-Time Polymerase Chain Reaction ; Transfection ; Up-Regulation

Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs.
Animals ; Antibodies, Viral ; blood ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Classical swine fever virus ; genetics ; Female ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Haemophilus parasuis ; genetics ; Immunization ; Interferon-gamma ; metabolism ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics

This study was conducted to investigate the expression of three genes related to early embryonic development in bovine transgenic cloned embryos. To accomplish this, development of bovine transgenic somatic cell nuclear transfer (SCNT) embryos was compared with non-transgenic embryos. Next, mRNA transcription of three specific genes (DNMT1, Hsp 70.1, and Mash2) related to early embryo development in transgenic SCNT embryos was compared between transgenic and non-transgenic SCNTs, parthenogenetic embryos, and in vitro fertilization (IVF) embryos. Transgenic SCNT embryos showed significantly lower rates of development to the blastocyst stage than non-transgenic ones. To investigate normal gene expression, RNA was extracted from ten blastocysts derived from parthenogenesis, IVF, non-transgenic, and transgenic SCNT embryos and reverse-transcribed to synthesize cDNA. The cDNA was then subjected to PCR amplification and semi-quantified. More DNMT1 mRNA was detected in the transgenic SCNT group than the other three groups. Hsp 70.1 mRNA was detected in the IVF embryos, while lower levels were found in SCNT and parthenogenetic embryos. Mash2 mRNA was present at the highest levels in transgenic SCNT embryos. In conclusion, the higher levels of methylation and lower protein synthesis after heat shock in the transgenic SCNT embryos expected based on our results may cause lower embryonic development.
Animals ; Animals, Genetically Modified/genetics ; Basic Helix-Loop-Helix Transcription Factors/*genetics/metabolism ; Cattle/embryology/*genetics ; DNA (Cytosine-5-)-Methyltransferase/*genetics/metabolism ; Embryo, Mammalian/embryology/metabolism ; Female ; Fertilization in Vitro ; *Gene Expression Regulation, Developmental ; HSP70 Heat-Shock Proteins/*genetics/metabolism ; Nuclear Transfer Techniques/veterinary ; Parthenogenesis ; Pregnancy ; RNA, Messenger/genetics/metabolism ; Transcription, Genetic

The purpose of this study was to verify that a combination of mild hyperthermia and docetaxel chemotherapy produces synergistic antitumor effects and to explore the action mechanisms of this treatment approach. The effects of docetaxel on the proliferation of cells from the estrogen receptor (ER)-positive human breast cancer cell line MCF-7 and the ER-negative human breast cancer cell line MDA-MB-453 were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and effective experimental concentrations of docetaxel were determined. The effects of mild hyperthermia plus docetaxel therapy on apoptosis rate in the MCF-7 and MDA-MB-453 human breast cancer cell lines were analyzed by using flow cytometry with Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The effects of these combined treatments on cell cycle progression in the MCF-7 and MDA-MB-453 human breast cancer cell lines were examined by using flow cytometry. The effects of these combined treatments on the expression of apoptosis-related proteins and proteins in the mitogen-activated protein kinase (MAPK) pathways were analyzed by using Western blotting. The effects of these combined treatments on the expression of the heat shock protein 70 (HSP70) and the multi-drug resistance (MDR) gene product P-glycoprotein (Pgp) were examined by using Western blotting. The results showed that the half-maximal inhibitory concentration (IC50) of docetaxel for MCF-7 and MDA-MB-453 cells was 19.57±1.12 and 21.64±2.31 μmol/L respectively. Mild hyperthermia with docetaxel therapy could increase apoptosis rate in the MCF-7 and MDA-MB-453 cells. Apoptosis rate in MCF-7 and MDA-MB-453 cells was increased from (23.66±3.59)% and (18.51±3.17)% in docetaxel treatment group to (47.12±6.73)% and (55.16±7.42)% in mild hyperthermia plus docetaxel group, indicating that the mild hyperthermia and docetaxel therapeutic approaches exhibited significant synergistic antitumor effects. Treatments of mild hyperthermia plus docetaxel induced G2/M cell cycle arrest in the MCF-7 and MDA-MB-453 cells. Western blotting demonstrated that proteins in the MAPK pathway were expressed at higher levels in docetaxel-treated cells following mild hypothermia than those in cells treated with docetaxel alone. As compared with blank control group, cells from the mild hyperthermia plus docetaxel group exhibited significantly decreased B-cell lymphoma 2 (Bcl-2) protein expression but slightly increased Bcl-2-associated X protein (Bax) expression. Western blotting results revealed that HSP70 and Pgp expression levels were significantly increased following mild hypothermia. It was concluded that treatments of mild hyperthermia plus docetaxel inhibited the proliferation of human breast cancer cells, promoted apoptosis of breast cancer cells, and produced synergistic antitumor effects.
ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Breast Neoplasms ; metabolism ; Cell Cycle ; drug effects ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; Hot Temperature ; Humans ; MAP Kinase Signaling System ; MCF-7 Cells ; Receptors, Estrogen ; genetics ; metabolism ; Taxoids ; pharmacology

Cigarette smoke is associated with the development of several diseases, such as chronic obstructive pulmonary disease (COPD). The purpose of this study was to investigate genotoxicity and heat shock protein 70 (Hsp70) in human airway smooth muscle cells (HASMCs) exposed to cigarette smoke extract (CSE). HASMCs was exposed to CSE with different doses for 24 h. The level of 8-hydroxydeoxyguanosine (8-OHdG) was determined by using HPLC-ECD, the DNA damage was analyzed by using comet assay, and apoptosis was examined by using Annexin-FITC/PI staining. The production of Hsp70 after CSE stimulation was tested. Results indicated that CSE significantly increased the level of 8-OHdG, DNA damage and cell apoptosis, and reduced the production of Hsp70. In particular, levels of Hsp70 were inversely correlated with 8-OHdG, DNA damage and cell apoptosis. It was concluded that cigarette smoke induced genotoxicity and decreased the production of cell protective protein Hsp70, which may contribute to the development of some airway diseases.
Apoptosis ; DNA Damage ; Deoxyguanosine ; analogs & derivatives ; metabolism ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Lung ; cytology ; drug effects ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Smoke ; adverse effects ; Tobacco ; toxicity ; Tumor Cells, Cultured

OBJECTIVETo investigate HSP70 gene expression from Dendrobium officinale under low temperature stress, which will provide the molecular biological foundation for breeding the low temperature resistant strain.

METHODHSP70 gene full length cDNA was cloned by rapid amplification of cDNA ends (RACE) on the basis of HSP70 gene fragment sequences, and the structure and function of HSP70 gene were deduced. The expression of HSP70 under low temperature stress was detected by RT-PCR.

RESULTThe full length of HSP70 gene cDNA was 2 296 bp containing a 1 944 bp open reading frame (ORF) that encoded a protein of 647 amino acids. Its amino acids sequence had typical HSP70 characteristics and high homology with other plant's HSP70. Cold stress expression analysis showed that expression of the HSP70 gene could be induced by low temperature.

CONCLUSIONThe HSP70 gene of D. officinale was successfully cloned and reported for the first time which proved that the expression could be induced by low temperature. The cloning of HSP70 gene provides a stable foundation for further study of D. officinale cultivation and the breeding of the cold resistance strains.

Amino Acid Sequence ; Cloning, Molecular ; Cold Temperature ; Dendrobium ; classification ; genetics ; metabolism ; HSP70 Heat-Shock Proteins ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Plant Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment