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

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

Tea is a popular beverage. Recently, green tea was reported to increase the number of peroxisomes in rats. In this study, to find out whether the green tea-induced proliferation of peroxisomes is mediated by PPARalpha , a transient transfection assay was carried out to investigate the interactions of tea extracts (green tea, black tea,oolong tea and doongule tea) and tea components (epigallocatechin gallate, epigallocatechin, epicatechin gallate, epicatechin and gallic acid), with mouse cloned PPARalpha . Green tea and black tea extracts, and epigallocatechin gallate, a major component of fresh green tea leaves, increased the activation of PPAalpha 1.5-2 times compared with the control. It is suggested that the green tea induced-peroxisomal proliferation may be mediated through the transactivation of PPARalpha and that epigallocatechin gallate may be an effective component of green tea leaves. This would account for the increase in the number of peroxisomes and the activity of peroxisomal enzymes previously reported. However, black tea, a fully fermented product, had a stronger effect than oolong tea extract. These results also suggest, that in addition to epigallocatechin gallate, green tea leaves may possess some active chemicals newly produced as a result of the fermentation process, which act on PPARalpha like other peroxisome proliferators.
Animals ; COS Cells/enzymology ; Camellia sinensis ; Catechin/*analogs&derivatives/pharmacology ; Cercopithecus aethiops ; PPAR alpha/*metabolism ; Plant Extracts/*pharmacology ; Plasmids ; *Tea ; Trans-Activation (Genetics)/drug effects ; Transfection/veterinary

BACKGROUNDTo investigate the synergetic transactivating effects of HCV core and HBV X proteins.

METHODSHCV core and HBV X protein-expressing plasmids were constructed with the vector pcDNA3.1(-). The plasmids were transfected into HepG2 cells and cotransfected Hep2 cells with reporter plasmid Psv-lacZ by lipofectamine plus reagents. The virus proteins produced in transient expression system were detected at the transcription and translation levels. The activity of b-galactosidase was detected, which reflected the transactivating function of the proteins.

RESULTSThe expression of plasmids were detected in soluble protein cell extracts of transiently transfected HepG2 cells. HCV core protein activated the b-galactosidase expression at a value 4.9 times higher than the control, while HBV X protein activated at a value 3.5 times. It arrived at 9 times transfected with the plasmids simultaneously. The activating effect increased in relation to the amount of plasmids.

CONCLUSIONSThe results suggested that the two kinds of virus proteins have transactivating effect on SV40 early promoter/enhancer, and they acted synergistically. These contribute to explain the mechanisms of liver injury or tumorigenesis induced by HCV or/and HBV infection.

Animals ; Carcinoma, Hepatocellular ; virology ; Enhancer Elements, Genetic ; Hepacivirus ; genetics ; Hepatitis C Antigens ; genetics ; Humans ; Liver Neoplasms ; virology ; Promoter Regions, Genetic ; drug effects ; Simian virus 40 ; genetics ; Trans-Activators ; genetics ; Transcriptional Activation ; Viral Core Proteins ; genetics ; beta-Galactosidase ; biosynthesis ; genetics

OBJECTIVETo investigate the effect of EBV immediate-early protein Zta on cell cycle of Daudi cells and the involved mechanisms.

METHODSThe expression vector encoding Zta was constructed and electroporated into Daudi cells. Flow cytometric analysis was used to detect the cell cycle, Western blot to the protein levels of p21, Rb and E2F-1.

RESULTSThe vector was constructed successfully, the expression of Zta protein inhibited the proliferation of Daudi cells and promoted cell cycle from G(0)/G(1) phase \[(30.0 ± 3.4)%\] to S phase \[(47.7 ± 1.1)%\]. Meanwhile, Rb expression was significantly downregulated, E2F-1 and p21 expression upregulated by Zta.

CONCLUSIONZta could promote G(0)/G(1) phase to S phase transition in Daudi cells, which might be associated with the reduced expression of Rb and increased expression of E2F-1 and p21 protein.

Cell Cycle ; genetics ; Cell Division ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; E2F1 Transcription Factor ; metabolism ; Genetic Vectors ; Herpesvirus 4, Human ; genetics ; Humans ; Immediate-Early Proteins ; genetics ; Retinoblastoma Protein ; metabolism ; Trans-Activators ; genetics ; Transcriptional Activation ; Viral Proteins ; genetics

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.
Animals ; DNA-Binding Proteins ; analysis ; genetics ; Electrophoretic Mobility Shift Assay ; Luminescent Measurements ; NF-kappa B ; analysis ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Trans-Activators ; analysis ; genetics ; Transcription, Genetic ; Transcriptional Activation

American ginseng (AG) has been demonstrated to inhibit breast cancer cell growth in vitro. p21 protein, a universal cell cycle inhibitor, binds cyclin-CDK complexes, an important mechanism in cell cycle regulation. The purpose of this investigation was to determine if AG induces p21 gene expression in hormone sensitive (MCF-7) and insensitive (MDA-MB-231) breast cancer cell lines. Cells grown in steroid stripped medium (SSM) were treated with AG, 17-beta-estradiol (E2), genistein or cycloheximide (CHX). Northern blot analyses were performed using human p21Cip1 and 36B4 cDNA probes. Cell lines were transiently transfected with select mouse p21 CAT reporter constructs, including those lacking a p53 binding site. Cell cycle analyses was performed by FACScan. The results revealed that AG induced p21 mRNA expression in MCF-7 and MDA-MB-231 cells (p=0.0004; p< or =0.0001, respectively). Neither E2 nor genistein alter p21 mRNA expression. CHX, a protein synthesis inhibitor, did not block p21 mRNA expression induced by AG, indicating that p21 is induced as an immediate early gene. AG activated p21 reporter constructs in transfected cells, independent of p53 binding sites. The cell cycle proliferative phase was significantly decreased by AG and increased by E2 (p< or =0.0001). AG may inhibit breast cancer cell growth by transcriptional activation of the p21 gene, independent of p53.
Animal ; Binding Sites ; Breast Neoplasms ; Cell Division/drug effects ; Chloramphenicol O-Acetyltransferase/genetics ; Cyclins/*genetics ; Female ; Genes, Reporter ; HT29 Cells ; Human ; Mice ; *Panax ; Plant Extracts/pharmacology ; Protein p53/metabolism ; *RNA, Messenger ; *Trans-Activation (Genetics) ; Tumor Cells, Cultured

Animals ; Calcium ; metabolism ; DNA-Binding Proteins ; metabolism ; Enzyme Activation ; Focal Adhesion Kinase 2 ; metabolism ; Gene Expression Regulation, Viral ; Hepatitis B virus ; genetics ; physiology ; Humans ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Virus Replication

Mouse embryonic stem (mES) cells are capable of undergoing chondrogenesis in vitro. To enhance this process, the human SOX9 (hSOX9) cDNA was delivered into mES cells and the clones overexpressing hSOX9 (denoted as mES-hSOX9 cells) were verified by Western blot analysis. The transcripts of collagen IIA (a juvenile form), aggrecan and Pax1 were expressed in mES-hSOX9 cells grown on feeder layers, suggesting the immediate effect of exogenous SOX9 on chondrogenesis. However, SOX9 overexpression did not affect the cell cycle distribution in undifferentiated mES cells. Upon differentiation, collagen IIB (an adult form) was detected in day 3 immature embryoid bodies. In addition, the overexpression of exogenous SOX9 significantly induced transcriptional activity driven by SOX9 binding site. Taken together, we for the first time demonstrated that constitutive overexpression of exogenous SOX9 in undifferentiated mES cells might have dual potentials to induce both chondrogenic commitment and growth capacity in the undifferentiated status.
Animals ; Cell Differentiation/genetics ; Cell Line ; *Chondrogenesis ; Collagen Type II/genetics ; Embryo/*cytology ; Enhancer Elements (Genetics)/genetics ; Extracellular Matrix Proteins/genetics ; Genetic Markers/genetics ; High Mobility Group Proteins/genetics/*metabolism ; Humans ; Lectins, C-Type/genetics ; Mice ; Paired Box Transcription Factors/genetics ; Proteoglycans/genetics ; Research Support, Non-U.S. Gov't ; Stem Cells/*metabolism/physiology ; Trans-Activation (Genetics) ; Transcription Factors/genetics/*metabolism

We examined the effect of class II transactivator (CIITA) down-modulation on allograft rejection. To inhibit the function of CIITA, we constructed a series of CIITA mutants and found one exhibiting the dominant-negative effect on the regulation of major histocompatibility complex (MHC) class II expression. To test whether the CIITA dominant-negative mutant reduces immunogenecity, CIITA-transfected melanoma cells were injected into allogeneic host and assessed for immune evading activity against host immune cells. We demonstrated that the CIITA dominant-negative mutant allowed tumor nodules to develop earlier in the lung than control by this tumor challenge study. Furthermore, skin grafts deficient for CIITA also survived longer than wild-type in allogeneic hosts. Both the tumor challenge and skin graft studies suggest the inhibition of CIITA molecules in donor tissue would be beneficial to the control of allo-response.
Transplantation, Homologous ; Transfection ; Trans-Activators/genetics/*immunology/metabolism ; Trans-Activation (Genetics)/genetics/immunology ; Skin Transplantation ; Nuclear Proteins/genetics/*immunology/metabolism ; Mutation ; Mice, Transgenic ; Mice, Knockout ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Melanoma, Experimental/genetics/immunology/pathology ; Male ; Interferon Type II/pharmacology ; Humans ; Histocompatibility Antigens Class II/genetics/*immunology/metabolism ; Graft Survival/genetics/immunology ; Graft Rejection/genetics/*immunology ; Genes, MHC Class II/genetics/immunology ; Flow Cytometry ; DNA, Complementary/genetics ; Cell Proliferation/drug effects ; Cell Line, Tumor ; Animals