Objective: To investigate the expression characteristic of the Daxx gene in the mouse testis and its role in spermatogenesis. METHODS: Real-time PCR, Western blot and immunofluorescence were used in examining the expression characteristics of DAXX in the testis tissue from wild-type, Sertoli cell-specific androgen receptor knockout (SCARKO) and androgen receptor knockout (ARKO) mice at different postnatal weeks . RESULTS: The Daxx gene was highly expressed in the testis tissue and mainly in the nuclei of the wild-type mice at 4 postnatal weeks. Compared with the wild-type, the ARKO mice showed a markedly decreased expression of DAXX (0.299±0.026), which displayed a polar distribution in the spermatogenic cells (0.853±0.058) and exhibited no significant difference in the SCARKO mice (1.000±0.015). CONCLUSIONS The Daxx gene expression is the highest in the middle-stage development of the mouse testis, significantly decreased in ARKO mice as compared with the wild-type, and its location influenced by specific AR knockout in Sertoli cells. DAXX may be involved in the regulation of spermatogenesis in mice.
Animals ; Carrier Proteins ; genetics ; metabolism ; Cell Nucleus ; genetics ; metabolism ; Gene Expression ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Knockout ; Molecular Chaperones ; Nuclear Proteins ; genetics ; metabolism ; Receptors, Androgen ; genetics ; Sertoli Cells ; Spermatogenesis ; genetics ; Testis ; metabolism

To analyze the differentially expressed proteins which interacted with NF-kappaB in the uterine lower segment smooth muscle tissues under different status of labor onset, and to provide a new foundation on the mechanisms for labor onset.
 Methods: NF-κB P65 protein expression in smooth muscle tissues from the term non-labor group, natural term labor group and drug-induced term labor group was analyzed by Western blot. Co-immunoprecipitation and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) were performed to detect the proteins interacting with NF-κB p65 in the NF-κB p65 complexes. The components of the complex were identified by LC-ESI-MS/MS (liquid chromatography-tandem electrospray mass spectrometry) and database analysis. The identified differentially expressed proteins were confirmed by Western blot.
 Results: Positive expression of NF-κB was detected in all of the three groups. 10 differentially expressed proteins were identified by LC-ESI-MS/MS in human lower segment myometrium tissues in the term non-labor group and natural term labor group, mean while, 5 differentially expressed proteins were identified in the term non-labor group and the drug-induced labor group. 3 differential expression proteins were detected in all of the 3 groups, including Heat shock 70, Annexin A6 and Desmin, which were verified by Western blot. These proteins were mainly involved in chaperone, signal transduction, cell structure, and energy metabolism process, respectively.
 Conclusion: NF-κB expressed in uterine smooth muscle cells is involved in the process of initiation and regulation of labor onset through a number of proteins relevant to signal transduction, cell structure and energy metabolism.
Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Energy Metabolism ; genetics ; Female ; Humans ; Immunoprecipitation ; Labor, Obstetric ; genetics ; Molecular Chaperones ; genetics ; Myocytes, Smooth Muscle ; Myometrium ; physiology ; NF-kappa B ; genetics ; physiology ; Pregnancy ; Protein Interaction Mapping ; Proteomics ; Signal Transduction ; genetics ; Tandem Mass Spectrometry ; Transcription Factor RelA

Azacitidine ; pharmacology ; Base Sequence ; Chromosomes, Human, X ; genetics ; DNA Methylation ; drug effects ; DNA Mutational Analysis ; Galactosyltransferases ; metabolism ; Gene Expression Regulation ; drug effects ; Glomerulonephritis, IGA ; etiology ; genetics ; metabolism ; Glycosylation ; Humans ; Immunoglobulin A ; metabolism ; Lipopolysaccharides ; pharmacology ; Lymphocytes ; metabolism ; Molecular Chaperones ; genetics ; metabolism ; Mutation ; Polymorphism, Single Nucleotide

As a large family of hydrolases, GTPases are widespread in cells and play the very important biological function of hydrolyzing GTP into GDP and inorganic phosphate through binding with it. GTPases are involved in cell cycle regulation, protein synthesis, and protein transportation. Chaperones can facilitate the folding or refolding of nascent peptides and denatured proteins to their native states. However, chaperones do not occur in the native structures in which they can perform their normal biological functions. In the current study, the chaperone activity of the conserved GTPases of Escherichia coli is tested by the chemical denaturation and chaperone-assisted renaturation of citrate synthase and α-glucosidase. The effects of ribosomes and nucleotides on the chaperone activity are also examined. Our data indicate that these conserved GTPases have chaperone properties, and may be ancestral protein folding factors that have appeared before dedicated chaperones.
Citrate (si)-Synthase ; chemistry ; Cloning, Molecular ; Conserved Sequence ; Escherichia coli ; cytology ; enzymology ; GTP Phosphohydrolases ; chemistry ; genetics ; isolation & purification ; metabolism ; Guanosine Diphosphate ; pharmacology ; Guanosine Triphosphate ; analogs & derivatives ; pharmacology ; Molecular Chaperones ; chemistry ; genetics ; isolation & purification ; metabolism ; Protein Denaturation ; drug effects ; Protein Renaturation ; drug effects ; Ribosomes ; metabolism ; alpha-Glucosidases ; chemistry

OBJECTIVESTo investigate the function and possible mechanisms of PIAS3 expression on the invasion of TJ905 cells.

METHODSPIAS3 overexpression vectors were constructed and PIAS3 siRNA were chemically synthesized, which were separately transfected into TJ905 cells for upregulation or downregulation of PIAS3 expression levels in TJ905 cells. After that, the invasive effects of TJ905 cells were measured by Transwell assay, and the expression of PIAS3, tissue inhibitor of metalloproteinases (TIMP)3, matrix metalloprotease (MMP)-2, and MMP-9 were identified by Western blot.

RESULTSIn vitro transfection efficiency of plasmids and oligonucleotides were separately 85.3% ± 3.1% and 95.1% ± 2.9%. PIAS3 overexpression plasmid transfection in vitro could effectively improve the expression of PIAS3 protein in TJ905 cells and inhibit the invasion of TJ905 cells (P < 0.05), and cell penetration ratio reduced from 87.9% ± 9.3% to 37.3% ± 7.9% compared with control group, while it upregulated TIMP3 and downregulated MMP-2, MMP-9 protein expression (P < 0.05); PIAS3 siRNA transfection could inhibit the PIAS3 protein expression of TJ905 cells and promote the invasion of TJ905 cells (P < 0.05), and cell penetration ratio increased from 83.9% ± 7.1% to 93.2% ± 3.1% compared with control group, while it downregulated TIMP3 and upregulated MMP-2, MMP-9 protein expression (P < 0.05).

CONCLUSIONPIAS3 expression is closely related to the invasion properties of glioma TJ905 cells.

Cell Line, Tumor ; Genetic Vectors ; Glioma ; metabolism ; pathology ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Molecular Chaperones ; genetics ; metabolism ; Neoplasm Invasiveness ; Protein Inhibitors of Activated STAT ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Tissue Inhibitor of Metalloproteinase-3 ; metabolism ; Transfection

Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release (IICR) via IP(3)R(1), but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an IICR model, Ca(2+) image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP(3)Rs (IICR) and the IICR were markedly decreased in ERp44 overexpressed Hela cells. The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331-377) mutants of ERp44 on IICR were significantly decreased compared with ERp44. However, the binding capacity of ERp44 to L3V domain of IP(3)R(1) (1L3V) was enhanced by ERp44 C160S/C212S mutation. Taken together, these results suggest that the mutants of ERp44, C160/C212, can more tightly bind to IP(3)R(1) but exhibit a weak inhibition of IP(3)R(1) channel activity in Hela cells.
Adenosine Triphosphate ; pharmacology ; Amino Acid Substitution ; Biological Transport ; drug effects ; physiology ; Blotting, Western ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; physiology ; HeLa Cells ; Humans ; Immunoprecipitation ; Inositol 1,4,5-Trisphosphate ; metabolism ; Inositol 1,4,5-Trisphosphate Receptors ; physiology ; Membrane Potentials ; drug effects ; physiology ; Membrane Proteins ; genetics ; metabolism ; Microscopy, Confocal ; Molecular Chaperones ; genetics ; metabolism ; Mutation ; Plasmids ; Transfection

Dendritic-cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN; CD209) has an important role in mediating adherence of Mycobacteria species, including M. tuberculosis and M. bovis BCG to human dendritic cells and macrophages, in which these bacteria can survive intracellularly. DC-SIGN is a C-type lectin, and interactions with mycobacterial cells are believed to occur via mannosylated structures on the mycobacterial surface. Recent studies suggest more varied modes of binding to multiple mycobacterial ligands. Here we identify, by affinity chromatography and mass-spectrometry, four novel ligands of M. bovis BCG that bind to DC-SIGN. The novel ligands are chaperone protein DnaK, 60 kDa chaperonin-1 (Cpn60.1), glyceraldehyde-3 phosphate dehydrogenase (GAPDH) and lipoprotein lprG. Other published work strongly suggests that these are on the cell surface. Of these ligands, lprG appears to bind DC-SIGN via typical proteinglycan interactions, but DnaK and Cpn60.1 binding do not show evidence of carbohydrate-dependent interactions. LprG was also identified as a ligand for DC-SIGNR (L-SIGN; CD299) and the M. tuberculosis orthologue of lprG has been found previously to interact with human toll-like receptor 2. Collectively, these findings offer new targets for combating mycobacterial adhesion and within-host survival, and reinforce the role of DCSIGN as an important host ligand in mycobacterial infection.
Amino Acid Sequence ; Bacterial Adhesion ; physiology ; Bacterial Proteins ; genetics ; metabolism ; Cell Adhesion Molecules ; genetics ; metabolism ; Chromatography, Affinity ; Dendritic Cells ; metabolism ; microbiology ; Host-Pathogen Interactions ; genetics ; physiology ; Humans ; In Vitro Techniques ; Lectins, C-Type ; genetics ; metabolism ; Ligands ; Macrophages ; metabolism ; microbiology ; Mass Spectrometry ; Membrane Proteins ; genetics ; metabolism ; Models, Biological ; Molecular Chaperones ; genetics ; metabolism ; Molecular Sequence Data ; Mycobacterium bovis ; genetics ; metabolism ; Mycobacterium tuberculosis ; genetics ; metabolism ; pathogenicity ; Pulmonary Surfactant-Associated Protein A ; metabolism ; Receptors, Cell Surface ; genetics ; metabolism

When we treated rat bone marrow stromal cells (rBMSCs) with neuronal differentiation induction media, typical unfolded protein response (UPR) was observed. BIP/GRP78 protein expression was time-dependently increased, and three branches of UPR were all activated. ATF6 increased the transcription of XBP1 which was successfully spliced by IRE1. PERK was phosphorylated and it was followed by eIF2alpha phosphorylation. Transcription of two downstream targets of eIF2alpha, ATF4 and CHOP/GADD153, were transiently up-regulated with the peak level at 24 h. Immunocytochemical study showed clear coexpression of BIP and ATF4 with NeuN and Map2, respectively. UPR was also observed during the neuronal differentiation of mouse embryonic stem (mES) cells. Finally, chemical endoplasmic reticulum (ER) stress inducers, thapsigargin, tunicamycin, and brefeldin A, dose-dependently increased both mRNA and protein expressions of NF-L, and, its expression was specific to BIP-positive rBMSCs. Our results showing the induction of UPR during neuronal differentiations of rBMSCs and mES cells as well as NF-L expression by ER stress inducers strongly suggest the potential role of UPR in neuronal differentiation.
Activating Transcription Factor 4/genetics/metabolism ; Animals ; Apoptosis/drug effects ; Bone Marrow Cells/*cytology ; Cell Differentiation ; Culture Media/pharmacology ; Embryonic Stem Cells/*cytology ; Endoplasmic Reticulum/genetics/metabolism ; Gene Expression/drug effects ; Heat-Shock Proteins/*genetics/metabolism ; Mice ; Microtubule-Associated Proteins/genetics/metabolism ; Molecular Chaperones/*genetics/metabolism ; Nerve Tissue Proteins/genetics/*metabolism ; Neurofilament Proteins/genetics/metabolism ; Neurons/*cytology ; Nuclear Proteins/genetics/metabolism ; Protein Folding ; Rats ; Stromal Cells

OBJECTIVEPseudomonas aeruginosa is a ubiquitous and opportunistic pathogen that uses the type III secretion system (TTSS) to inject effector proteins directly into the cytosol of target cells to subvert the host cell's functions. Specialized bacterial chaperones are required for effective secretion of some effectors. To identify the chaperone of ExoS, the representative effector secreted by the TTSS of P. aeruginosa, we analyzed the role of a postulated chaperone termed Orf1.

METHODSBy allelic exchange, we constructed the mutant with the deletion of gene Orf1. Analysis of secreted and cell-associated fractions was performed by SDS-PAGE and Western blotting. Using strain expressing in trans Orf1, tagged by V5 polypeptide and histidine, protein-protein interaction was determined by affinity resin pull-down assay in combination with MALDI-TOF. The role of Orf1 in the expression of exoS was evaluated by gene reporter analysis.

RESULTSPull-down assay showed that Orf1 binds to ExoS and ExoT. Secretion profile analysis showed that Orf1 was necessary for the optimal secretion of ExoS and ExoT. However, Orf1 had no effect on the expression of exoS.

CONCLUSIONOrf1 is important for the secretion of ExoS probably by maintaining ExoS in a secretion-competent conformation. We propose to name Orf1 as SpcS for "specific Pseudomonas chaperone for ExoS".

ADP Ribose Transferases ; genetics ; metabolism ; Bacterial Toxins ; genetics ; metabolism ; Base Sequence ; Blotting, Western ; DNA Primers ; Electrophoresis, Polyacrylamide Gel ; Kinetics ; Molecular Chaperones ; genetics ; metabolism ; Protein Binding ; Pseudomonas aeruginosa ; metabolism ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

As thermostable enzymes and organisms are much more needed, researches on heat shock proteins(HSPs) of hyperthermophilic archaea have drawn more concerns. HSPs from hyperthermophilic archaea are concise only with HSP60, sHSP, prefoldin and AAA+proteins, but without HSP100s, HSP90s, HSP70 (DnaK), HSP40 (DnaJ) and GrpE which are common in mesophilic or thermophilic archaea. Accordingly, studies on the structure, function and operation mechanism of these four groups are much more important and meaningful. This review focuses on the recent progress in the researchs on the structure, function, operation mechanism and cooperation of the HSPs from hyperthermophilic archaea. The problems and obfuscations in these HSPs are analyzed, and farther research direction and key points are put out.
Archaea ; classification ; metabolism ; Archaeal Proteins ; metabolism ; Chaperonin 60 ; metabolism ; Heat-Shock Proteins ; genetics ; metabolism ; Molecular Chaperones ; metabolism