OBJECTIVE: To investigate the effects of total flavonids of astragalus(TFA) on arrhythmia, endoplasmic reticulum stress and connexcin in mice with viral myocarditis and to clarify the mechanisms of TFA against viral myocarditis complicated with arrhythmia. METHODS: Thirty-six male Balb/c mice were randomly divided into control group, viral myocarditis group and total flavonoids group (=12). The mice of viral myocarditis were intraperitonealy injected with 0.1 ml/day 10-950 TCID CVB3 for 3 days. The mice of TFA group were intraperitoneal injected with 0.1 ml/day 10-950 TCID CVB3 for 3 days and treated with 0.1ml, 20 mg/L TFA by tail vein injection. At the end of the experiment, arrhythmia was detected by electrocardiogram, the heart of mice were stained by HE, the expressions of glucose-regulated protein 78(GRP78), endoplasmic reticulum stress signaling pathway factor activating transcription factor 4(ATF4) and connexcin 43(Cx43) were detected by Western blot. RESULTS: The expressions of GRP78 and ATF4 were increased and the expression of Cx43 was decreased in viral myocarditis, while TFA inhibited these effect of viral myocarditis in heart of mice. CONCLUSIONS The antiarrhythmic effect of TFA may be related to the alleviation of endoplasmic reticulum stress and the increase of Cx43 expression.
Activating Transcription Factor 4 ; metabolism ; Animals ; Arrhythmias, Cardiac ; drug therapy ; Astragalus Plant ; chemistry ; Connexin 43 ; metabolism ; Coxsackievirus Infections ; drug therapy ; Drugs, Chinese Herbal ; pharmacology ; Endoplasmic Reticulum Stress ; drug effects ; Flavonoids ; pharmacology ; Heat-Shock Proteins ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; virology ; Myocardium

Protein palmitoylation, one of post-translation modifications, refers to the addition of saturated 16-carbon palmitic acid to cysteine residues via the thioester bond. It plays key roles in various functional activities, such as the interaction, stability and location of proteins. Heat shock protein 90 (Hsp90), an important molecular chaperone, has been reported to be involved in sperm capacitation. However, it remains unclear whether protein palmitoylation exists in sperm and whether Hsp90 in sperm is palmitoylated under different physiological conditions. In this study, we examined whether the protein palmitoylation is present in mouse cauda epididymis sperm using acyl-biotin exchange method, predicted the potential palmitoylated sites of Hsp90 by the software CSS-Palm 4.0 and detected the palmitoylated Hsp90 in the mouse sperm from caput epididymis and cauda epididymis by immunoprecipitation. We found that some proteins, approximately 50, 65, 72, 85 and 130 kDa, were palmitoylated in mouse cauda epididymis sperm. Five sites in two Hsp90 isoforms were predicted to be palmitoylated. The results also showed that Hsp90 in mouse sperm was palmitoylated and its palmitoylation level was involved in different physiological conditions: the palmitoylation level of cauda epididymis sperm was higher than that of caput epididymis sperm; and the palmitoylation level after capacitation was much higher than that before capacitation. In conclusion, this study reveals that protein palmitoylation is present in mouse sperm and the palmitoylated Hsp90 is associated with different physiological conditions in sperm.
Animals ; Epididymis ; HSP90 Heat-Shock Proteins ; metabolism ; Lipoylation ; Male ; Mice ; Palmitic Acid ; chemistry ; Sperm Capacitation ; Spermatozoa ; metabolism

OBJECTIVETo investigate the effect of total flavonoids of astragalus on the expression of endoplasmic reticulum chaperone, calumenin and connecxin 43 (CX43) in suckling mouse myocardium with myocarditis caused by coxsackievirus B3 (CVB3).

METHODSThe primary culture of suckling mouse myocardium cells were randomly divided into control group, CVB3 infected group and total flavonoids of astragalus group. Firstly, to confirm the identity of the suckling mouse myocardium, α-SMA was monitored by immunohistochemistry method. Then the protein expression changes of endoplasmic reticulum chaperone-glucose regulatory protein 78 ( GRP78), calumenin and CX43 were detected by Western blot.

RESULTS(1) Compared with that of the control group, the GRP78 expression level in CVB3 infected group was improved, the expression levels of calumenin and CX43 were all reduced. (2) Compared with that of CVB3 infected group, GRP78 expression level was decreased, and the expression levels of calumenin and CX43 were increased in total flavonoids of astragalus group.

CONCLUSIONCVB3 infection may cause endoplasmic reticulum stress of rat myocardium cells by increasing the expression of GRP78 and decreasing the expression of calumenin and CX43. On the other hand, total flavonoids of astragalus can reduce the expression of GRP78 and increase the expression of calumenin and CX43.The results of this experiment may be closely related to the effects of anti-arrhythmia with viral myocarditis caused by CVB3.

Animals ; Astragalus Plant ; chemistry ; Blotting, Western ; Calcium-Binding Proteins ; metabolism ; Cells, Cultured ; Connexin 43 ; metabolism ; Coxsackievirus Infections ; drug therapy ; Endoplasmic Reticulum ; metabolism ; Endoplasmic Reticulum Stress ; drug effects ; Flavonoids ; pharmacology ; Heat-Shock Proteins ; metabolism ; Mice ; Myocarditis ; drug therapy ; virology ; Myocardium ; cytology ; Myocytes, Cardiac ; drug effects ; virology ; Rats

Mutations or inactivation of parkin, an E3 ubiquitin ligase, are associated with familial form or sporadic Parkinson's disease (PD), respectively, which manifested with the selective vulnerability of neuronal cells in substantia nigra (SN) and striatum (STR) regions. However, the underlying molecular mechanism linking parkin with the etiology of PD remains elusive. Here we report that p62, a critical regulator for protein quality control, inclusion body formation, selective autophagy and diverse signaling pathways, is a new substrate of parkin. P62 levels were increased in the SN and STR regions, but not in other brain regions in parkin knockout mice. Parkin directly interacts with and ubiquitinates p62 at the K13 to promote proteasomal degradation of p62 even in the absence of ATG5. Pathogenic mutations, knockdown of parkin or mutation of p62 at K13 prevented the degradation of p62. We further showed that parkin deficiency mice have pronounced loss of tyrosine hydroxylase positive neurons and have worse performance in motor test when treated with 6-hydroxydopamine hydrochloride in aged mice. These results suggest that, in addition to their critical role in regulating autophagy, p62 are subjected to parkin mediated proteasomal degradation and implicate that the dysregulation of parkin/p62 axis may involve in the selective vulnerability of neuronal cells during the onset of PD pathogenesis.
Adaptor Proteins, Signal Transducing ; chemistry ; metabolism ; Animals ; HEK293 Cells ; Heat-Shock Proteins ; chemistry ; metabolism ; Humans ; Lysine ; metabolism ; Mice ; Neurons ; metabolism ; pathology ; Oxidopamine ; pharmacology ; Parkinson Disease ; metabolism ; pathology ; Proteasome Endopeptidase Complex ; metabolism ; Protein Stability ; Proteolysis ; drug effects ; Sequestosome-1 Protein ; Ubiquitin-Protein Ligases ; metabolism ; Ubiquitination ; drug effects

BACKGROUND/AIMS: Most pesticide formulations contain both chief and additive ingredients. But, the additives may not have been tested as thoroughly as the chief ingredients. The surfactant, nonyl phenoxypolyethoxylethanol (NP40), is an additive frequently present in pesticide formulations. We investigated the effects of NP40 and other constituents of a validamycin pesticide formulation on cell viability and on the expression of genes involved in cell damage pathways. METHODS: The effects of validamycin pesticide ingredients on cell viability and of NP40 on the mRNA expression of 80 genes involved in nine key cellular pathways were examined in the human neuroblastoma SK-N-SH cell line. RESULTS: The chemicals present in the validamycin pesticide formulation were cytotoxic to SK-N-SH cells and NP40 showed the greatest cytotoxicity. A range of gene expression changes were identified, with both up- and down-regulation of genes within the same pathway. However, all genes tested in the necrosis signaling pathway were down-regulated and all genes tested in the cell cycle checkpoint/arrest pathway were up-regulated. The median fold-change in gene expression was significantly higher in the cell cycle checkpoint/arrest pathway than in the hypoxia pathway category (p = 0.0064). The 70 kDa heat shock protein 4 gene, within the heat shock protein/unfolded protein response category, showed the highest individual increase in expression (26.1-fold). CONCLUSIONS: NP40 appeared to be particularly harmful, inducing gene expression changes that indicated genotoxicity, activation of the cell death (necrosis signaling) pathway, and induction of the 70 kDa heat shock protein 4 gene.
Aged ; Cell Cycle Checkpoints/drug effects/genetics ; Cell Line, Tumor ; Cell Survival/drug effects ; Dose-Response Relationship, Drug ; Female ; Gene Expression Regulation/drug effects ; Genes, cdc ; HSP110 Heat-Shock Proteins/genetics/metabolism ; Humans ; Inositol/*analogs & derivatives/chemistry/poisoning ; Necrosis ; Neurons/*drug effects/metabolism/pathology ; Nonoxynol/chemistry/*toxicity ; Pesticides/chemistry/*poisoning ; RNA, Messenger/metabolism ; Signal Transduction/drug effects ; Surface-Active Agents/chemistry/*toxicity

Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 (MYH9), heat shock cognate protein 70 (Hsc70), and dnaJ homolog subfamily A member 1 (DNAJA1), were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.
Acetylation ; Actins ; chemistry ; metabolism ; Animals ; Cell Line ; Chromatography, High Pressure Liquid ; HSC70 Heat-Shock Proteins ; metabolism ; HSP40 Heat-Shock Proteins ; metabolism ; Histone Deacetylase 6 ; Histone Deacetylases ; metabolism ; Isotope Labeling ; Liver ; metabolism ; Lysine ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Microscopy, Confocal ; Nonmuscle Myosin Type IIA ; metabolism ; Protein Binding ; Proteomics ; Substrate Specificity ; Tandem Mass Spectrometry

OBJECTIVETo explore the mechanism of podophyllotoxin nanostructured lipid carriers (POD-NLC)-induced apoptosis of VK2/E6E7 cells mediated by endoplasmic reticulum stress (ERS).

METHODSVK2/E6E7 cells cultured in vitro were exposed to 0.125, 0.25, and 0.5 µg/ml POD-NLC or blank NLC for 24 h. The intracellular calcium concentration was measured by laser scanning confocal microscopy (LSCM), and the expression levels of GRP78, GRP94, and calpain2 mRNA and proteins in the cells were detected using RT-PCR and Western blotting.

RESULTSCompared with the control cells, the cells exposed to POD-NLC showed a concentration-dependent increase of intracellular calcium concentration (P<0.01), and the differences were statistically significant between different dose groups (P<0.05). RT-PCR and Western blotting showed that POD-NLC up-regulated GRP78, GRP94 and calpain2 mRNA and proteins expressions, which showed significant differences between blank-NLC and the control groups (P>0.05).

CONCLUSIONPOD-NLC induces apoptosis of VK2/E6E7 cells possibly by triggering the endoplasmic reticulum stress response.

Apoptosis ; Calcium ; metabolism ; Calpain ; metabolism ; Cell Line ; Endoplasmic Reticulum Stress ; HSP70 Heat-Shock Proteins ; metabolism ; Heat-Shock Proteins ; metabolism ; Humans ; Membrane Proteins ; metabolism ; Nanostructures ; chemistry ; Podophyllotoxin ; chemistry ; RNA, Messenger

OBJECTIVETo explore the effect of Se-riched soybean peptide (SSP) on antioxidant function in rats of fatty liver caused by high-fat diet.

METHODSForty Wistar rats were divided into 4 groups randomly and fed with standard diet and water (NC), high-fat diet and water (HC), high-fat diet and SSP (0.1 g/d) (SeH), standard diet and SSP (0.1 g/d) (SeN) respectively. After 10 weeks, the rats were killed to investigate the pimelosis level in liver tissues by Sudan III staining and the expression of hepatic GRP78 by immunohistochemical analysis. We also analyzed the changes of liver function, blood lipid, the glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in livers and serum.

RESULTSThe pimelosis level, total cholesterol (TC), triglyceride (TG), MDA contents and the expression of GRP78 in HC group were significantly higher than those in NC, SeN, SeH groups. The activities of GSH-Px and SOD in liver and serum were markedly up-regulated in SeH (P < 0.01). There was no significant difference between NC and SeN groups.

CONCLUSIONSSP can improve liver cell injury and the antioxidant functions in rats with fatty liver effectively and decrease the expression of GRP78 in liver.

Animals ; Antioxidants ; metabolism ; Diet, High-Fat ; adverse effects ; Disease Models, Animal ; Fatty Liver ; chemically induced ; metabolism ; Heat-Shock Proteins ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Rats ; Rats, Wistar ; Selenium ; pharmacology ; Soybean Proteins ; pharmacology ; Soybeans ; chemistry

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

In the present study, a new compound named 17-(6-cinnamamido-hexylamino-)-17-demethoxygeldanamycin (CDG) was obtained by introducing the cinnamic acid (CA) group into the 17-site of geldanamycin (GDM). The anti-cancer effects of CDG in vitro and in vivo were evaluated. MTT assay was used to examine the inhibitory effect of CDG on the proliferation of MCF-7, HepG2, H460 and SW1990 cells. Immunofluorescent staining flow cytometry combined with Annexin V-FITC/PI staining were used to detect apoptotic cells. Transwell assay was used to analyze the effect of CDG on cell invasion and migration ability. Western blotting was used to detect the expression levels of RAF-1, EGFR, AKT, CDK4 and HER-2 of MCF-7, HepG2 and H460 cells. The toxicities of CDG and GDM were evaluated in mice. Using the subcutaneously transplanted MCF-7 xenograft in nude mice, inhibitory effect was evaluated in vivo. The results showed that CDG inhibited the proliferation of cancer cells (IC50: 13.6-67.4 microg.mL-1). After exposure to CDG for 48 h, most cells presented typical morphologic changes of apoptosis such as chromatin condensation or shrunken nucleus. The rates of apoptosis of MCF-7, HepG2, H460 and SW1990 cells incubated with 10 microg.mL-1 CDG were 23.16%, 27.55%, 22.21%, 20.47%, respectively. A dose-dependent reduction of migration of four cell lines was found after exposure to CDG. The decreased levels of RAF-1, EGFR, AKT, CDK4 and HER-2 showed that CDG possessed HSP90 inhibitory effect. The result of animal toxicity test on the mice suggested that CDG had lower toxicity than GDM. Meanwhile, CDG inhibited the growth of MCF-7 xenografts of athymic mice.
Animals ; Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Apoptosis ; drug effects ; Benzoquinones ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase 4 ; metabolism ; Female ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; chemical synthesis ; chemistry ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Proto-Oncogene Proteins A-raf ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Random Allocation ; Receptor, Epidermal Growth Factor ; metabolism ; Receptor, ErbB-2 ; metabolism ; Tumor Burden ; drug effects ; Xenograft Model Antitumor Assays