Apoptosis ; Hepatocytes ; pathology ; Humans ; Trans-Activators ; physiology

OBJECTIVETo construct a subtractive cDNA library of genes transactivated by hepatitis B virus X protein (HBX) using suppression subtractive hybridization (SSH) technique and to clone genes associated with HBX transactivating function.

METHODSThe mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-X and pcDNA3.1(-) empty vector respectively, then cDNA was synthesized. After restriction enzyme RsaI digestion, a number of small size cDNA was obtained. Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor. After tester cDNA was hybridized with driver cDNA twice and underwent nested polymerase chain reaction (PCR) twice the production was subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was carried out with E. coli strain JM109, some cDNA was sequenced and analyzed in GenBank with Blast.

RESULTSThe subtractive cDNA library of genes transactivated by HBX was constructed. The amplified library contained 85 positive clones, and colony PCR showed that these clones contained 200-1000 bp inserts. 65 clones were analyzed by sequencing and bioinformatics, which suggested nineteen known genes and fifteen genes with unknown function.

CONCLUSIONA subtractive cDNA library of genes transactivated by HBX using SSH technique has been constructed successfully, which may bring some new clues for studying the biological functions of HBX and the pathogenesis of hepatoma.

Cloning, Molecular ; Gene Library ; RNA, Messenger ; analysis ; Trans-Activators ; physiology ; Transcriptional Activation

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China, which is mainly caused by hepatitis B virus (HBV) infection. The X gene product (HBx) of HBV has extensive trans-activating functions. HBx affects the signal transduction, apoptotic cell death and cell cycle through interaction with variety intracellular proteins in infected hepatocytes. In view of the importance of HBx in HBV replication and in hepatic cell functions, the role of HBx in HCC development has been attracting great attention.
Carcinoma, Hepatocellular ; pathology ; Hepatitis B virus ; genetics ; physiology ; Humans ; Liver Neoplasms ; pathology ; Trans-Activators ; genetics ; physiology ; Virus Replication ; physiology

In order to study function of NAC transcription in development, hormone regulation and the stress response of Salvia miltiorrhiza, the NAC transcription was cloned and analyzed. By retrieving cDNA database of S. miltiorrhiza hairy root one NAC unigene was found, then a full length of cDNA was cloned by designing specific primers and PCR amplifying. Using ORF finder it was found that the cDNA containing a NAC-AB conserved domain in N-terminal, so the cDNA was a NAC transcription factor, named as SmNAC1 (kF006346). Bioinformatics analysis showed that SmNAC1 had an open reading frame (ORF) of 591 bp encoding 196 amino acids. The calculated protein had isoelectric point (pI) of 4.36 with molecular weight about 21.66 kDa. The transcription level of SmNAC1 after dealing with yeast extract (YE) and silver ion (Ag+) in S. miltiorrhiza hairy root was markedly stimulated up regulating. It was 1.4 fold compared with the control after induction 2 h, and maintained 2.0 fold on 4-12 h after induction. SmNAC1 may participate in regulation of stress response of YE + Ag+.
Cloning, Molecular ; Computational Biology ; Phylogeny ; Plant Proteins ; genetics ; physiology ; Plant Roots ; chemistry ; Salvia miltiorrhiza ; chemistry ; genetics ; Trans-Activators ; genetics ; physiology

PURPOSETo review the recent developments in the structure and function of Smad proteins.

DATA SOURCESBoth Chinese- and English-language literatures were searched using MEDLINE/CD-ROM (1997 - 2000) and the Index of Chinese-Language Literature (1997 - 2000).

STUDY SELECTIONData from published articles about TGF-beta signal transduction in recent domestic and foreign literature were selected.

DATA EXTRACTIONData were mainly extracted from 22 articles which are listed in the reference section of this review.

RESULTSSmad proteins mediate signal transduction induced by the TGF-beta superfamily. Based on their structural and functional properties, Smad proteins are divided into three groups. The first group, receptor-regulated Smads (R-Smads), are phosphorylated by activated type I receptors and form heteromeric complexes with the second group of Smads, common mediator Smads (Co-Smads). These Smad complexes translocate into the nucleus to influence gene transcription. Inhibitory Smads (I-Smads) are the third group and these antagonize the activity of R-Smads. In the nucleus, Smads can directly contact Smad-binding elements (SBE) in target gene promoters. Through interaction with different transcription factors, transcriptional co-activators or co-repressors, Smads elicit different effects in various cell types. The aberrance of Smad proteins has been noted in several human disorders such as fibrosis, hypertrophic scarring and cancer.

CONCLUSIONThe structure of Smads determines their function as transcriptional factors which translocate signals from the cell surface to the nucleus where Smads regulate TGF-beta superfamily-dependent gene expression.

DNA-Binding Proteins ; chemistry ; physiology ; Humans ; Signal Transduction ; Smad Proteins ; Trans-Activators ; chemistry ; physiology ; Transcription Factors ; physiology ; Transforming Growth Factor beta ; physiology

OBJECTIVETo investigate whether hepatitis B x protein (HBx) stimulates vascular endothelial growth factor (VEGF) through hypoxia inducible factor-1 (HIF-1 alpha) pathway.

METHODSTwo plasmids including pIRES-EGFP-HBx and pTK-Hyg were co-transfected to a hepatocellular carcinoma cell line SMMC-7721. With fluorescence-positive and fluorescence-negative hygromycin-resistant colonies selected, expressions of VEGF and HIF-1 alpha in protein or/and mRNA level were detected.

RESULTSFluorescence-positive cells were stably integrated with HBx, in which expression of HIF-1 alpha and VEGF were upregulated. Fluorescence-negative cells did not express HBx, VEGF or HIF-1 alpha.

CONCLUSIONHBx can activate VEGF through HIF-1 alpha pathway.

Cell Line, Tumor ; Gene Expression Regulation ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; Trans-Activators ; physiology ; Transcription Factors ; genetics ; physiology ; Vascular Endothelial Growth Factor A ; genetics ; physiology

Carcinoma, Hepatocellular ; etiology ; DNA, Viral ; metabolism ; Hepatitis B ; complications ; Hepatitis C ; complications ; Humans ; Liver Neoplasms ; etiology ; Trans-Activators ; physiology ; Viral Core Proteins ; physiology ; Viral Nonstructural Proteins ; physiology

The most important factor in the pathogenesis of biomaterial-associated Staphylococcal infections is the formation of bacterial biofilms. Biofilm formation was regulated or influenced by quorum sensing. One of the quorum sensing systems agr is genus specific which controls the expression of a series of toxins and virulence factors and the interaction with the innate immune system. New research indicates that the role of agr during infection is controversial. The research progress will play an important role in the development of novel antibacterial agents and management of device-related infection of Staphylococci.
Bacterial Proteins ; physiology ; Biocompatible Materials ; Biofilms ; growth & development ; Gene Expression Regulation, Bacterial ; Humans ; Signal Transduction ; genetics ; Staphylococcal Infections ; microbiology ; Staphylococcus ; genetics ; physiology ; Trans-Activators ; physiology

Confocal laser scanning microscopy was used to observe the spatio-temporal expression of the pathway-specific gene redD during S. coelicolor cell cultivation. The corresponding mutant S. coelicolor lyqRY1522 carrying redD::eyfp in the chromosome was constructed. The temporal expression results of the fusion protein during submerged cultivation demonstrated that expression of redD began in the transition phase, continuing through the exponential growth phase to the stationary phase, and reached maximum in the stationary phase. On the other hand, redD was expressed only in substrate mycelia during solid-state culture, while aerial mycelia remained essentially non-fluorescent throughout culture. Results demonstrated that the expression pattern of redD coincides with that of the biosynthesis of the antibiotics during culture, revealing a direct correlation between the spatio-temporal distribution of regulatory gene expression and second metabolism.
Anti-Bacterial Agents ; biosynthesis ; Bacterial Proteins ; genetics ; metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Bacterial ; physiology ; Mutation ; Signal Transduction ; physiology ; Streptomyces coelicolor ; genetics ; metabolism ; Trans-Activators ; genetics ; metabolism

Animals ; Glucose ; pharmacology ; Homeodomain Proteins ; analysis ; genetics ; physiology ; Immunohistochemistry ; Insulin ; secretion ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Trans-Activators ; analysis ; genetics ; physiology