Specific mutations of basal core promoter are associated with chronic liver disease in hepatitis B virus subgenotype D1 prevalent in Turkey

The role of hepatitis B virus (HBV) genetics in the clinical manifestations of infection is being increasingly recognized. Genotype D is one of eight currently recognized major HBV genotypes. The virus is ubiquitous worldwide, but shows different features in different regions. One hundred and ninety‐eight patients with chronic HBV infection were enrolled in this study, 38 of whom had been diagnosed with cirrhosis of the liver and/or hepatocellular carcinoma. HBV DNA was isolated from the patients' blood samples and the entire genome and/or the basal core promoter/core promoter region sequenced. Phylogenetic analysis of the complete genomes revealed that subgenotype D1 is the most prevalent subgenotype in Turkey, but there was no definite phylogenetic grouping according to geography for isolates from different regions within Turkey, or for isolates in Turkey relative to other parts of the world. Turkish isolates tended to be genetically similar to European and central Asian isolates. Overall, HBV‐infection in Turkey appears to be characterized by early HBeAg seroconversion, a high incidence of the A1896 core promoter mutation and a small viral load. Genotype D characteristic mutations A1757 and T1764/G1766 were found in the BCP region. T1773 was associated with T1764/G1766 and a larger viral load. In conclusion, infection with HBV genotype D in Turkey has a similar clinical outcome to that of Europe and central Asia. Genotypic mutations in genotype D may be linked with disease prognosis in Turkey, but further studies with higher sample numbers and balanced clinical groups are needed to confirm this.     

乙型肝炎病毒的核心启动子各区段功能的研究

将一系列核心启动子区的缺失突变引入乙肝病毒（ＨＢＶ）线性转录单元,从病毒的抗原,ＲＮＡ以及子代ＤＮＡ等各个水平,分析了各缺失突变对前基因组ＲＮＡ和前核心ＲＮＡ转录的影响,对核心启动子各片段的功能进行了深入的研究。Ｃ片段缺失后检测不到ｅ抗原和前核心ＲＮＡ,却仍有核心抗原和前基因组ＲＮＡ的合成以及病毒子代ＤＮＡ的复制;而Ｂ３片段缺失后ｅ抗原和核心抗原均有显著下降,但仍能检测到两种ｍＲＮＡ的合成和病毒子     

乙型肝炎病毒核心启动子下游克隆入Teto序列后其在不同细胞系中转录活性的变化

目的:探讨将Teto序列克隆入乙型肝炎病毒核心启动子(Cp)下游后对该启动子在不同细胞系中转录活性的影响。方法:利用PCR从质粒pTL-8中扩增7个Teto序列,并将其克隆入T载体pMD19-T/Simple中,测序正确后将7个Teto序列亚克隆入pGL3-Basic/Cp萤光素酶报告基因质粒中Cp启动子的下游,以构建质粒pGL3-Basic/Cp/x7t。将能表达TetR的质粒pTL-8的萤光素酶编码基因切除后,与pGL3-Basic/Cp/x7t共转染肝癌细胞系HepG2、宫颈癌细胞系HeLa、绿猴肾细胞系COS-7、乳腺癌细胞系MDA-MB-231和结肠癌细胞系HT-29,通过双萤光素酶检测系统检测萤光素酶在这些不同细胞系中的活性,以分析将Teto序列克隆入乙型肝炎病毒核心启动子下游后对Cp在不同细胞系中转录活性的影响。结果:构建了质粒pGL3-Basic/Cp/x7t,将其转染HeLa、COS-7、HepG2、MDA-MB-231和HT-29细胞后其相对萤光素酶活性分别为56.14、171.52、211.03、6.11和34.24。结论:将Teto序列克隆入Cp下游并不能明显提高Cp的转录活性,而且破坏了Cp的组织特异性转录活性。     

Structural variants of the vitamin D analogue EB1089 reduce its ligand sensitivity and promoter selectivity

The nuclear hormone 1α,25-dihydroxyvitamin D₃ (VD) has important cell-regulatory functions but also a strong calcemic effect. Therefore, various VD analogues have been synthesized and screened for their biological profile. In order to gain more insight into the molecular basis of the high antiproliferative but low calcemic action of the VD analogue EB1089, we characterized this compound in comparison to five structurally related VD analogues. The activities of the six VD analogues in in vitro assays (limited protease digestion assays for determining interaction with monomeric vitamin D receptor (VDR), ligand-dependent gel shift assays for showing the increase of DNA binding of VDR-retinoid X receptor (RXR) heterodimers, and reporter gene assays on different types of VD response elements for demonstrating the efficacy in nuclear VD signalling) were found to represent their biological potency (antiproliferative effect on different malignant cell lines). In this series, EB1089 proved to be the most potent VD analogue; that is, every structural modification (20-epi configuration, cis-configuration at position C24, or changes at the ethyl groups at position C25) appeared to reduce the determined activities mediated through the VDR of these analogues. Moreover, the modifications of EB1089 resulted in a loss of VD response element selectivity, suggesting that this parameter is very critical for the biological profile of this VD analogue. J. Cell. Biochem. 71:340–350, 1998. © 1998 Wiley-Liss, Inc.     

1α,25-Dihydroxyvitamin D3 receptor as a mediator of transrepression of retinoid signaling

The receptors for retinoic acid (RA) and for 1α,25-dihydroxyvitamin D₃ (VD), RAR, RXR, and VDR are ligand-inducible members of the nuclear receptor superfamily. These receptors mediate their regulatory effects by binding as dimeric complexes to response elements located in regulatory regions of hormone target genes. Sequence scanning of the tumor necrosis factor-α type I receptor (TNFαRI) gene identified a 3′ enhancer region composed of two directly repeated hexameric core motifs spaced by 2 nucleotides (DR2). On this novel DR2-type sequence, but not on a DR5-type RA response element, VD was shown to act through its receptor, the vitamin D receptor (VDR), as a repressor of retinoid signalling. The repression appears to be mediated by competitive protein–protein interactions between VDR, RAR, RXR, and possibly their cofactors. This VDR-mediated transrepression of retinoid signaling suggests a novel mechanism for the complex regulatory interaction between retinoids and VD. J. Cell. Biochem. 67:287–296, 1997. © 1997 Wiley-Liss, Inc.     

Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II,depolarization and protein kinase C

The integrative nuclear FGFR1 signaling (INFS) pathway functions in association with cellular growth, differentiation, and regulation of gene expression, and is activated by diverse extracellular signals. Here we show that stimulation of angiotensin II (AII) receptors, depolarization, or activation protein kinase C (PKC) or adenylate cyclase all lead to nuclear accumulation of fibroblast growth factor 2 (FGF-2) and FGFR1, association of FGFR1 with splicing factor-rich domains, and activation of the tyrosine hydroxylase (TH) gene promoter in bovine adrenal medullary cells (BAMC). The up-regulation of endogenous TH protein or a transfected TH promoter-luciferase construct by AII, veratridine, or PMA (but not by forskolin) is abolished by transfection with a dominant negative FGFR1TK-mutant which localizes to the nucleus and plasma membrane, but not by extracellularly acting FGFR1 antagonists suramin and inositolhexakisphosphate (IP6). Mechanism of TH gene activation by FGF-2 and FGFR1 was further investigated in BAMC and human TE671 cultures. TH promoter was activated by co-transfected HMW FGF-2 (which is exclusively nuclear) but not by cytoplasmic FGF-1 or extracellular FGFs. Promoter transactivation by HMWFGF-2 was accompanied by an up-regulation of FGFR1 specifically in the cell nucleus and was prevented FGFR1(TK-) but not by IP6 or suramin. The TH promoter was also transactivated by co-transfected wild-type FGFR1, which localizes to both to the nucleus and the plasma membrane, and by an exclusively nuclear, soluble FGFR1(SP-/NLS) mutant with an inserted nuclear localization signal. Activation of the TH promoter by nuclear FGFR1 and FGF-2 was mediated through the cAMP-responsive element (CRE) and was associated with induction of CREB- and CBP/P-300-containing CRE complexes. We propose a new model for gene regulation in which nuclear FGFR1 acts as a mediator of CRE transactivation by AII, cell depolarization, and PKC.     

反向斑点杂交法检测HBV基本核心启动子区A1762T／G1764A突变的实验研究

目的建立一种基于尼龙膜的反向斑点杂交法,用于检测乙型肝炎病毒（HBV）基本核心启动子区（BCP）A1762T／G1764A突变。方法根据我国HBV主要流行的基因型为B和C,从GenBank上查出4种HBVBCP序列。利用在线工具ClustalW进行比对,针对该突变位点设计引物和检测探针。探针经合成和修饰后点在带正电的尼龙膜上。将反向斑点杂交法结合地高辛检测试剂盒用于检测A1762T／G1764A突变,以测序法确定该区域序列的标本为检测对象。结果反向斑点杂交法分别检测5例A1762／G1764病毒株、2例T1762／G1764病毒株、5例A1762／A1764病毒株和4例T1762／A1764病毒株,结果与测序完全相同。结论应用本方法可以快速、准确地HBV相关的热点突变。     

Critical role of helix 12 of the vitamin D(3) receptor for the partial agonism of carboxylic ester antagonists.

The carboxy-terminal alpha-helix of a nuclear receptor ligand-binding domain (LBD), helix 12, contains a critical, ligand-modulated interface for the interaction with coactivator proteins. In this study, using the example of the vitamin D receptor (VDR) and the partial antagonist ZK159222, the role of helix 12 (residues 417-427) for both antagonistic and agonistic receptor actions was investigated. Amino acid residue G423 was demonstrated to be critical for partial agonism of ZK159222, but not for the activity of the natural VDR agonist, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)). The amount of partial agonism of ZK159222 increased when helix 12 was truncated by the last four amino acid residues (Delta424-27) and augmented even more, when in addition helix 12 of VDR's dimerization partner, retinoid X receptor (RXR), was truncated. In contrast, the low agonism of a structural derivative of ZK159222, ZK168281, was not affected comparably, whereas other close structural relatives of ZK159222 even demonstrated the same agonistic activity as that of 1alpha,25(OH)(2)D(3). The amount of agonism of ZK159222 and ZK168281 at different variations of helix 12 correlated well with VDR's ability to complex with coactivator proteins and inversely correlated with the strength of the compound's antagonistic action on 1alpha,25(OH)(2)D(3) signalling. Molecular dynamics simulations of the LBD complexed with the two antagonists could explain their different action by demonstrating a more drastic displacement of helix 12 through ZK168281 than through ZK159222. Moreover, the modelling could indicate a kink of helix 12 at amino acid residue G423, which provides the last four amino acid residues of helix 12 with a modulatory role for the partial agonism of some VDR antagonists, such as ZK159222. In conclusion, partial agonism of a VDR antagonist is lower the more it disturbs helix 12 in taking the optimal position for coactivator interaction.