Receptor activation of curcumin on human peroxisome proliferators-activated receptors γ1 / 中草药

Objective: To determine whether curcumin is a natural ligand for human peroxisome proliferators-activated receptors γ1 (hPPARγ1) by measuring the combination ability and internal activity. Methods: The combination ability was determined by radioactively labeled ligand binding experiment (RBCA), and the internal activity was estimated by trans-activation reporter gene test. Results: The combination ability of curcumin on hPPARγ1 showed that IC50 was (8.82 ± 0.74) μmol/L, and Ki was 0.72 μmol/L. The internal activity showed that EC50 was 7.3 μmol/L and Emax was 43.3. Conclusion: Curcumin has affinity and intrinsic activity with hPPARγ1, which suggests that curcumin may be a natural ligand of hPPARγ1.

Mechanisms of STAT family mediated IDO regulation in dendritic cells / 国际肿瘤学杂志

Indoleamine 2,3-dioxygenase (IDO) is known as an endogenous immunosuppressive enzyme which plays a significant role in the process of tumor.IDO is not only found in tumor cells but also detected in dendritic cell (DC) in tumor microenvironment,which participates in the formation of tumor immune tolerance through expressing IDO enzyme.Signal transducer and activator of transcription (STAT) is the main signal protein family which participates in the IDO transcriptional regulation of DC.It is necessary to detail the signaling pathway in regulating IDO expression,which will help us develop high specific and more active IDO inhibitors and provide new options for anti-cancer targeted therapy.

Hypoxia activates the cyclin D1 promoter via the Jak2/STAT5b pathway in breast cancer cells

Hypoxia, a common consequence of solid tumor growth in breast cancer or other cancers, serves to propagate a cascade of molecular pathways which include angiogenesis, glycolysis, and various cellcycle control proteins. As we have shown previously, hypoxia activates STAT5 (signal transducer and activator of transcription 5) and increases its binding activity to the GAS element in mammary epithelial cells. In this study we attempted to elucidate the mechanism by which cyclin D1 is regulated by the STAT5 protein under hypoxic conditions. Our data demonstrate that hypoxia (2% O2) or desferrioxamine (DFO) induces tyrosine and serine phosphorylation of STAT5 in human breast cancer cells (MCF-7) and mammary epithelial cells (HC11). Imunoprecipitation and subsequent Western analysis showed that Jak2 leads to the tyrosine phosphorylation and activation of STAT5a or STAT5b under hypoxic conditions. Using a transfected COS-7 cell model system, we demonstrate that the activity of a cyclin D1 promoter-luciferase construct increased under hypoxic conditions or DFO treatment. The activity of the STAT5b/cyclin D1 promoter increased significantly by 12 h of hypoxia, whereas the activity of the STAT5a/cyclin D1 promoter was unaffected under hypoxic conditions. These increases in promoter activity are predominantly mediated by the Jak2/ STAT5b signaling pathway. We have shown by EMSA that hypoxia induces STAT5 to bind to the cyclin D1 promoter (GAS-1) in MCF-7 and HC11 cells. These data suggest that STAT5b may mediate the transcriptional activation of cyclin D1 after hypoxic stimulation.

Prokaryotic expression and bioinformatics analysis of NS5A transactivating protein 7 of hepatitis C virus / 解放军医学杂志

Objective To construct prokaryotic expression vector of NS5A transactivating protein 7 of hepatitis C virus (NS5ATP7), induce the expression of NS5ATP7 in E. coli, and to predict its structure and function by bioinformatics analysis. Methods NS5ATP7 gene was amplified by reverse transcription PCR (RT-PCR) using HepG2 cells mRNA as template, and was ligated into pGEM-T cloning vector. After verifying the sequence of the inserted fragment by restriction enzyme digestion identification and sequencing, NS5ATP7 was cloned into inducible prokaryotic expression vector pET-32a(+) and transfected into competent E. coli BL21. The protein expression was induced with IPTG and the product was analyzed by SDS-PAGE and Western blotting hybridization. The structure and function of NS5ATP7 were predicted using bioinformatics techniques. Results NS5ATP7 gene with about 891bp was amplified by RT-PCR, which was coincident with that we expected, and it was then inserted into expression vector pET-32a(+). Under the induction of IPTG, the recombinant NS5ATP7 was expressed successfully. SDS-PAGE and Western blotting assay showed that this recombinant protein possessed good immunogenicity. Bioinformatics method such as ExPASy, TMHMM and SignalP analysis indicated that NS5ATP7 was full of helices, had neither transmembranous structure nor signal peptide. Conclusions The recombinant NS5ATP7 gene could be successfully expressed in prokaryotic expression system of E. coli, which might lay the foundation of studying the immunogenicity and biological charactersitics of the NS5ATP7. Bioinformatics analysis may provide a new method to analyze the structure and function of a new protein.

Screening and cloning of the target genes transactivated by HCV FTP2 protein using suppression subtractive hybridization technique / 中华传染病杂志

Objective To clone and identify human genes transactivated by homo sapiens HCV FTP2 by constructing a cDNA subtractive library with suppression subtractive hybridization tech- nique.Methods Suppression subtractive hybridization(SSH)and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HCV FTP2.The mRNA was iso- lated from HepG2 cells transfected pcDNA3.1(-)-HCV FTP2 and pcDNA3.1(-)empty vector re- spectively,and SSH method was employed to analyze the differentially expressed DNA sequence be- tween the two groups.After digestion with restriction enzyme Rsa I,small-size cDNAs were ob- tained.Then tester cDNA was divided into two groups and ligated to the specific adaptor I and adap- tor 2 respectively.After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR and then was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E.coli strain DH5?.Futhermore,the cDNA was se- quenced and analyzed in GenBank with Blast search after PCR.Results The subtractive library of genes transactivated by HCV FTP2 was constructed successfully.The amplified library contains 71 positive clones.Colony PCR shows that 56 clones contain 200～1000 hp inserts.Sequence analysis was performed in 24 clones randomly,and the full length sequences were obtained with bioinformatics method.Altogether 20 coding sequences in total were obtained,consisting of 19 known and 1 un- known.Conclusion The obtained sequences may be target genes transactivated by HCV FTP2,and some genes coding proteins involved in cell cycle regulation,metabolism and cell apoptosis.

Up-regulation of thioredoxin reductase 1 gene promoter by hepatitis B virus pre-S2 protein / 解放军医学杂志

Objective To investigate the activity of HBV pre-S2 protein on thioredoxin reductase 1 (TXNRD1) gene promoter. Methods TXNRD1 gene promoter DNA sequence was identified in GenBank by bioinformatics and amplified from HepG2 genome by polymerase chain reaction (PCR). The amplified product was cloned into pCAT3-Basic reporter vector,named pCAT3-TXNRD1p. The HepG2 cells were transfected by pCAT3-TXNRD1p,and then co-tranfected by pCAT3-TXNRD1p and pcDNA3.1(-)-preS2 plasmids. The choloraphenical acetyltransferase(CAT)activity was assessed by enzyme linked immunosorbent assay(ELISA). Results The results indicate that HepG2 cells transfected by pCAT3-TXNRD1p had higher activity of CAT than that transfected by pCAT3-Basic. The expression of CAT in HepG2 cells co-transfected by pCAT3-TXNRD1p and pcDNA3.1(-)-preS2 was 2.2 times higher than that with pCAT3-TXNRD1p. Conclusions The TXNRD1 gene promoter identified in this study has transcription activity and HBV pre-S2 protein can transactivate the expression of TXNRD1 gene.

Suppression subtractive hybridization for cloning of genes transactivated by c-terminally truncated middle surface protein of hepatitis B virus / 中华传染病杂志

Objective To construct a cDNA subtractive library of genes transactivated by c-terminally truncated middle surface protein of hepatitis B virus(MHBs t) with suppression subtractive hybridization technique for cloning genes associated with transactivation. Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-Mt167 and pcDNA3.1(-) empty vectors, respectively, then cDNA was synthesized. After restriction enzyme Rsa I digestion, small-size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After tester cDNA was hybridized with driver cDNA twice and underwent two times of nested PCR and then was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. Results The subtractive library of genes transactivated by MHBs t was constructed successfully. The amplified library contained 94 positive clones. Colony PCR showed that these clones contained 200-800bp inserts. Sequence analysis was performed in 50 clones,and the full length sequences were obtained with bioinformatics method. 23 coding sequences were obtained in total, which consisted of 19 known and 4 unknown ones.Conclusions The obtained sequences may be target genes transactivated by MHBs t, among which some genes coding proteins may involve in cell cycle regulation, immune response and tumour genesis.

Genotype-specific functional differences of the hepatitis B virus X protein / 中华传染病杂志

pcDNA3.1/Hygro(-)+pCMV?. Conclusions Transactivation competence of genotype B HBx was higher than that of genotype C HBx, while the antiproliferative and apoptosis effects of genotype B HBx was lower than of genotype C HBx. B and C genotype-specific functional differences of HBx may closely co-related with the pathogenicity of HBV.

Cloning of human gene 5 trans-activated by pre-S1 protein of hepatitis B virus / 解放军医学杂志

Objective To clone a new human gene 5 trans-activated by pre-S1 protein of hepatitis B virus (HBV), PS1TP5, and explore its structure and function by bioinformatics analysis. Methods PS1TP5 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) technique by using HepG2 cDNA as template and inserted into pGEM-T vector by TA cloning. Recombinant eukaryotic expression vector pcDNA TM 3.1/myc-His A-PS1TP5 had been constructed by subcloning, followed by restriction enzyme digestion analysis and sequencing. Bioinformatic methods were used to analyze its possible physical and chemical characters, structure, and function. Results PS1TP5 was successfully amplified and cloned into pGEM-T and pcDNA TM 3.1/myc-His A vector by RT-PCR from HepG2 cDNA. The new gene had been confirmed by sequencing after PCR identification and restriction enzyme digestion and named as PS1TP5 because of its trans-active function. The sequence for the PS1TP5 gene had been deposited into GenBank, the accession number was AY427953. Bioinformatics analysis showed that its ORF was 438bp and translated a protein of 145 aa. Conclusion A new gene-PS1TP5 has been recognized, and its recombinant eukaryotic expression vector (pcDNA TM 3.1/myc-His A-PS1TP5) has been constructed. These results will certainly bring some new clues for the study of the biological function of new gene and pathogenesis of chronic hepatitis B.

Cloning and identification of human gene transactivated by hepatitis C virus core protein 1 / 解放军医学杂志

Objective To screen and clone the target genes transactivated by hepatitis C virus (HCV) core protein. Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-core and pcDNA3.1(-) empty vector, respectively, and suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequences of the two groups. The obtained sequences were searched for homologous DNA sequence from GenBank. One of them was confirmed to be a new gene without homology with known genes in this database.Then, electric polymerase chain reaction was conducted for the cloning of the full-length DNA of the new gene, in conjunction with Kozak rule and the existence of polyadenyl signal sequence. The reverse transcription PCR (RT-PCR) was used to amplify the new gene with mRNA from HepG2 cell as the template. The coding sequence for the new gene was deduced according to the nucleotide sequence. Results A new gene with unknown function was named TAHCCP1.The nucleotide sequence of the new gene and its corresponding protein-encoding amino acid, which was 2 001nt and composing 667aa, have been determined. The sequence of the TAHCCP1 gene has been registered in GenBank with its accession number AY038359. Conclusion TAHCCP1 gene transactivated by HCV core protein was cloned and identified successfully by a combination of molecular biological technology and bioinformatics technique. The results are expected to pave the way for the study of the molecular mechanism of the transactivating effects of HCV core protein and the development of new therapies for chronic hepatitis C.

MOLECULAR MECHANISM OF PATHOGENESIS OF HEPATITIS C VIRUS / 解放军医学杂志

The pathogenesis of hepatitis C virus (HCV) is different from that of DNA virus and retrovirus. The epitope inducing neutralizing antibody in high variable region 1 (HVR1) is the reason for escape of host immune surveillance, evolution of chronic infection, and also difficulty of producing wide spectrum vaccine. Mimotope selected by using phage display technique is an alternative to solve this problem. The study on the binding protein to non coding region of HCV RNA is helpful to uncover the regulation of replication and translation of HCV genome. A study of the specific receptor of HCV is important to elucidate the mechanism of how HCV enter the hepatocyte. Yeast two hybrid is a useful technique to study the protein protein interactions between HCV and hepatocyte. Suppressive subtraction hybridization is an efficient method for identification of genes of interest transactivated by HCV structural and non structural proteins. All these studies will finally resulted in the full understanding of HCV pathogenesis, and will supply unique opportunity for the development of a new therapy for HCV infection.

Studies on RNase H of hepatitis B virus DNA polymerase to up-regulate the expression of cellular apoptosis susceptibility gene / 解放军医学杂志

Objective To study the activation effect of HBV RNase H protein on the transcription of cellular apoptosis susceptibility gene (CAS). Methods The promoter of DNA sequence of CAS gene was identified in GenBank by bioinformatics and amplified from HepG2 genome by PCR using sense (5′-GGTACCCGATTACATGTTGTACATGAAGG-3′) and antisence (5′-CTCGAGGCTGAGTTCCATTGCTATAG-3′) primers. As these primers contained Kpn I and Xho I recognition sites on their respective 5′-ends, the amplified DNA fragments were tested by sequencing and then subcloned into Kpn I/Xho I sites of pCAT3-Basic reporter vector by routine molecular biological methods. The reconstructed plasmid named pCAT3-CASp was identified by enzyme digestion of Kpn I/Xho I, in which the expression of chloramphenical acetyltransferase (CAT) was under the control of the promoter of CAS. The HepG2 cells were transfected by pCAT3-CASp, and then co-transfected by pCAT3-CASp and pcDNA3.1(-)-RH plasmids. At the same time, the empty pCAT3-basic and pCAT3-TXNRD1p were transfected (self-contructed by the authors) as controls. After 24h culturing, cells were collected and the expression of CAT activity was detected by ELISA according to the manufacturer′s protocol. Results The optical density of expression of CAT of pCAT3-CASp was 0.043 by ELISA, in contrast, the optical density of expression of pCAT3-Basic was 0.024. The expression of CAT in co-transfection of pCAT3-CASp and pcDNA3.1(-)-RH(0.065) was 1.5 times as higher as pCAT3-CASp plasmid (0.043), and 2.7 times as higher as pCAT3-Basic. Conclusions The CAS gene promoter identified in present study has transcription activity and HBV RNase H protein may activate the expression of CAS gene.