Cardiovascular Diseases ; metabolism ; pathology ; Humans ; Smad Proteins ; metabolism ; Trans-Activators ; metabolism

OBJECTIVESTo study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1).

METHODSBoth HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo.

RESULTSBoth HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1.

CONCLUSIONSHBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.

Histone Deacetylase 1 ; metabolism ; Humans ; Hydroxamic Acids ; metabolism ; Immunoprecipitation ; Plasmids ; Protein Interaction Mapping ; Trans-Activators ; metabolism

YAP/TAZ are wild over-activated in head and neck squamous cell carcinoma (HNSCC) with high potential as a direct therapy target for HNSCC treatments. However, the efforts on the directly targeting-YAP/TAZ therapies over the past decade, have very limited impacts, mainly caused by: 1. There is still none effective and specific YAP/TAZ inhibitor with clinical potential; 2. YAP/TAZ might not be directly targeted, because of their multiple important biological functions, such as: regulation of cell proliferation and survival, stem cell maintain, regulation of organ development, organ size control, and tissue regeneration. Interestingly, the over-activation of YAP/TAZ in HNSCC mainly be regulated by upstream abnormal molecular or biological events, instead of genes alteration of YAP/TAZ. Therefore, exploring the alternative molecular events regulating YAP/TAZ activation and molecular mechanism in HNSCC might help to uncover novel indirect targets of YAP/TAZ therapies for HNSCC prevention and treatment.
Adaptor Proteins, Signal Transducing/metabolism* ; Head and Neck Neoplasms ; Humans ; Phosphoproteins/metabolism* ; Trans-Activators/metabolism* ; Transcription Factors

Transcriptional activators are required to turn on the expression of genes in a eukaryotic cell. Activators bound to the enhancer can facilitate either the recruitment of RNA polymerase II to the promoter or its elongation. This article examines a few selected issues in understanding activator functions and activation mechanisms.
Animals ; Humans ; Trans-Activators ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transcription, Genetic ; Transcriptional Activation

Cell Differentiation ; Cell Line, Tumor ; metabolism ; Cell Proliferation ; Humans ; Trans-Activators ; biosynthesis ; genetics ; Transfection

Infection of hepatitis B virus (HBV) is a main cause of liver diseases including hepatitis, cirrhosis and hepatocellular carcinoma (HCC). Among the HBV-encoded proteins, the HBV X protein (HBx) has been suspected to be strongly involved in HBV-associated liver pathogenesis. HBx, a virally encoded multifunctional regulator, has been shown to induce apoptosis, anti-apoptosis, proliferation, and transformation of cells depending on the cell lines, model systems used, assay protocols, and research groups. Among the several activities of HBx, the pro-apoptotic function of HBx will be discussed in this review. Given that the disruption of apoptosis pathway by HBx contributes to the liver pathogenesis, a better understanding of the molecular interference in the cellular pro-apoptotic networks by HBx will provide useful clues for the intervention in HBV-mediated liver diseases.
*Apoptosis ; Hepatitis B/etiology ; Liver Diseases/metabolism/virology ; Trans-Activators/*metabolism ; Tumor Necrosis Factors/metabolism ; Tumor Suppressor Protein p53/metabolism

To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-kappa B were labelled with DIG by terminal transferase. After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8% nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged. Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film. The results showed that nuclear proteins binded specifically to the NF-kappa B consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-kappa B in PMA group was more than that in PMA + PDTC group. It is suggested that detection of NF-kappa B by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories.
Chemiluminescent Measurements ; DNA-Binding Proteins/*analysis ; DNA-Binding Proteins/genetics ; Electrophoretic Mobility Shift Assay ; NF-kappa B/*analysis ; NF-kappa B/genetics ; NF-kappa B/metabolism ; Rats, Sprague-Dawley ; *Trans-Activation (Genetics) ; Trans-Activators/analysis ; Trans-Activators/genetics ; *Transcription, Genetic

OBJECTIVETo screen the hepatocyte proteins that interact with hepatitis B virus X protein (HBx).

METHODSThe recombinant plasmid pSos-HBx was constructed by inserting Sos-HBx fragment into the bait vector, and after sequence verification the plasmid was transformed into competent yeast cells. The expression and self-activation of Sos-HBx protein was detected in the yeast cells. The hepatocyte proteins interacting with the bait protein was screened with CytoTrap yeast two-hybrid technique.

RESULTSThe reconstructed plasmid harboring HBx gene expressed Sos-HBx protein in the yeast cells without self-activation of the protein. CytoTrap yeast two-hybrid system identified 6 hepatocyte proteins that interacted with HBx, including fibronectin 1, translationally controlled tumor protein, IQ motif and WD repeats 1, follistatin, orosomucoid 1, and disulfide isomerase family A member 3.

CONCLUSIONSix HBx-binding hepatocyte proteins have been identified using the CytoTrap yeast two-hybrid system, which provides clues for further investigation of the role of HBx protein in hepatitis and liver cancer.

Genetic Vectors ; Hepatocytes ; metabolism ; Humans ; Plasmids ; Protein Interaction Domains and Motifs ; Proteins ; metabolism ; Trans-Activators ; metabolism ; Two-Hybrid System Techniques

The objective of study was to investigate the relationship between expressions of CIITA and MHC molecules in five human cell lines. The expressions of MHC molecules and CIITA protein were detected by Western blot, immunohistochemistry and flow cytometry. The expression of CIITA gene was measured by RT-PCR. The results indicated that the expression of MHC-II molecules in 5 human cell lines was consistent with expression of CIITA. The cell lines constitutively expressed CIITA also expressed MHC-II molecules, the expression of MHC-II molecules in cell lines expressed CIITA after induction with IFN-gamma also recovered; the cell lines unexpressed CIITA after induction with IFN-gamma did not respond to IFN-gamma-promoting expression of MHC-II molecules. It is concluded that some cell lines cannot express MHC-II molecules which may be related with deficiency of CIITA expression. It suggest that CIITA participates in regulation of MHC-II molecule expression, which may plays a certain role in escape from carcinogenesis under surveillance of immune system.
Cell Line ; Genes, MHC Class II ; Humans ; Interferon-gamma ; pharmacology ; Nuclear Proteins ; metabolism ; Trans-Activators ; metabolism ; Tumor Cells, Cultured

OBJECTIVETo construct pEGFP-HBx eukaryotic expression plasmid and establish stable and effective transfected rat oval cell (LE/6) strain expressing EGFP-HBx fusion protein to explore the roles of HBx gene and oval cell in carcinogenesis of hepatocellular carcinoma (HCC).

METHODSHBx gene with EcoRI and Hind III endo-enzyme sites was obtained by using PCR from plasmid pcDNA3.1-HBx. The purified HBx gene fragment was inserted into pEGFP-N1 expression vector, and the recombinant plasmid pEGFP-HBx was identified by restriction endonuclease and DNA sequencing analysis. LE/6 cells were transfected with recombinant pEGFP-HBx by lipofectamine reagent. Resistant to G418 clones were selected, and expression of EGFP-HBx fusion protein in clones were examined directly with fluorescence microscope, and these clones were isolated and proliferated. The expression of HBx was detected by RT-PCR analysis and immunocytochemistry.

RESULTSPlasmid pEGFP-HBx has whole HBx gene base and correct reading frame as indicated by restriction endonuclease and DNA sequencing analysis. After transfecting with pEGFP-HBx plasmid, LE/6 cell clones expressing EGFP-HBx fusion protein were obtained. RT-PCR analysis and immunocytochemistry showed that HBx gene was only expression in transfected pEGFP-HBx cells.

CONCLUSIONSThe pEGFP-HBx recombinant expression vector was successfully constructed, and the stable transfected LE/6 strain expressing EGFP-HBx fusion protein was successfully established. It will be helpful in the further study on the roles of HBx and liver oval cell in carcinogenesis of HCC.

Animals ; Cell Line ; Genetic Vectors ; Hepatocytes ; cytology ; metabolism ; Plasmids ; genetics ; Rats ; Stem Cells ; cytology ; metabolism ; Trans-Activators ; genetics ; Transfection