Identification and characterization of a structural protein of hepatitis B virus: a polymerase and surface fusion protein encoded by a spliced RNA

The hepatitis B virus (HBV) genome is known to contain four conserved and overlapped open reading frames (ORFs) encoding the viral core, polymerase (P), surface (S), and X proteins. Whether HBV encodes other proteins has long been a major interest in the field. Using (32)P-labeling of an introduced protein kinase A site attached to the N- or C-terminus of the HBV polymerase gene, a 43-kDa P-S fusion protein was detected in cell lysate, secreted virions, and 22-nm subviral particles. Immunobiochemical studies showed that the 43-kDa protein contains the epitopes of the N-terminus of polymerase and most parts of the surface proteins. This 43-kDa protein was shown to be a glycoprotein, similar to the surface protein. RT-PCR and sequence analyses identified a spliced mRNA which was derived from pregenomic RNA with a deletion of 454 nucleotides (nt) from nt 2447 to 2902. This splice event creates a P-S fusion ORF. This finding is consistent with the result obtained from an immunobiochemical study. Mutations at the splice donor or acceptor site on the HBV genome abrogated the production of the 43-kDa protein. These mutants had no effect on viral replication in transfected HuH-7 cells. However, this P-S fusion protein is able to substitute for the LS protein in virion maturation. On the basis of these results, we conclude that the 43-kDa protein is a polymerase-surface fusion protein encoded by a spliced RNA. Similar to the LS protein, the 43-kDa P-S fusion protein is a structural protein of HBV and might play a role in the HBV life cycle.     

Congenital lactic acidosis: evaluation of the properties of the a199t natural variant of human pyruvate dehydrogenase e1alpha by in vitro mutation

One cause of congenital lactic acidosis is a mutation in the E1 alpha-subunit of the pyruvate dehydrogenase multienzyme complex. Little is known about the consequences of these mutations at the enzymatic level. Here we study the A199T mutation by expressing the protein in Escherichia coli. The specific activity is 25% of normal and the K(m) for pyruvate is elevated by 10-fold. Inhibitors of lactate dehydrogenase might be a useful therapy for patients with such mutations.     

Origin of the epaxial and hypaxial myotome in avian embryos

The myotome originates from the dermomyotome. Controversy surrounds the location of myotome precursor cells within the dermomyotome and their segregation from the dermomyotome. Here we addressed the problem of myotome formation by labeling dermomyotome cells using the quail-chick marking technique. We carried out five series of transplantation and replaced: (1) the medial third, (2) the intermediate third, (3) the lateral third, (4) the cranial half, (5) the caudal half of a thoracic dermomyotome. The grafting procedures were performed in HH-stages 15–17 of quail and chick embryos. The chimeras were reincubated for 2 days up to HH-stages 24–25. All of the grafted parts contributed to the myotome. The epaxial myotome is derived from the medial third of the dermomyotome, while the hypaxial myotome is formed by both the intermediate and lateral third of the dermomyotome. Ep- and hypaxial myotome domains meet in the thickest part of the myotome that is situated in the middle of its ventrolateral axis. Myotome growth in the epaxial domain begins earlier than in the hypaxial domain. Cranial and caudal edges of the dermomyotome contribute equally to both the epaxial and hypaxial myotomes. The first born myotome cells are located in the lateral part of the epaxial myotome and development then proceedes in medial and lateral directions. Accepted: 27 June 2000     

中学生对立违抗性障碍的父母养育方式和家庭环境特征的初步研究

目的：了解单纯对立违抗性障碍（ODD）儿童的父母养育方式及家庭功能。方法：应用向制儿童行为调查表、家庭环境量表中文版（FES～CV）、父母养育方式量表（EMBU），对115例单纯ODD儿童（研究组）和115名非ODD正常儿童（对照组）进行评定和病例对照分析，结果：ODD组儿童家庭矛盾性得分较对照组高（P〈0．01），ODD组父母双亲的“情感温暖．理解”得分均明显比对照组得分低（父亲P〈0．01，母亲P〈0.05）．而其“惩罚，严厉”和“拒绝，否认”二因子得分则明显比对照组高（P〈0．01）；ODD组母亲的“过分干涉，过度保护”，因子得分也明显高于对照组（P〈0．01）。结论：ODD中学生的家庭存在高度的矛盾性．他们的父母养育方式不良，应引起重视。     

Nucleolar localization of non-structural protein 3b, a protein specifically encoded by the severe acute respiratory syndrome coronavirus

The open reading frame 3 (ORF3) of the severe acute respiratory syndrome coronavirus (SARS-CoV) genome encodes a predicted 154-amino acid protein, which lacks similarities to any known protein, and is named 3b. In this study, it was shown that 3b protein was predominately localized to nucleus with EGFP tag at its N- or C-terminus. The localization patterns were similar in different transfected cells. Immuno-fluorescence assay revealed that 3b protein was co-localized well with C23 in nucleolus. C23, B23 and fibrillarin all are important nucleolar proteins, which localize in the region of the nucleolus. Co-transfection of p3b-EGFP with pC23-DsRed, pB23-DsRed and pfibrillarin-DsRed further confirmed 3b's nucleolus localization. With construction of serial truncated mutants of 3b, a region (residues 134-154 aa) responsible for nucleolar localization was determinated in 3b protein. These results provide a new insight for further functional studies of SARS-CoV 3b protein.     

葡萄糖和胆固醇对人肝细胞ANGPTL3基因和蛋白质表达的影响

目的 观察葡萄糖和胆固醇对人肝细胞株(L02)血管生成素样蛋白3(angiopoietin-like pro-tein3,ANGPTL3)表达的影响,探讨其与糖尿病和高胆固醇血症的关系.方法 以L02细胞为研究对象,将其分为葡萄糖组(G)和胆固醇组(CH).分别在葡萄糖浓度为5.6 mmol/L(G1)、7.0 mmol/L(G2)、11.1 mmol/L(G3)、28.0 mmol/L(G4)、33.0 mmol/L(G5)的培养液和含胆固醇10 μmol/L、25 μmol/L、50 μmol/L、100 μmol/L的培养液中培养.采用RT-PCR方法检测各组细胞ANGPTL3 mRNA表达量,用Western印迹方法检测其蛋白质表达水平.结果 G3～G5组葡萄糖可剂量依赖性地增加ANGPTL3的mRNA表达(P<0.05);在蛋白质表达水平,G2～G5组与G1组比较差异均有统计学意义(P<0.05);G1～G5组各组间两两比较,除G4组与G5组呈现表达量虽升高,但差异无统计学意义(P>0.05)外,其它各组间差异均有统计学意义(P<0.05).4种胆固醇浓度均可促进L02细胞ANGPTL3的mRNA和蛋白质表达(P<0.05).结论 在一定浓度范围内葡萄糖可剂量依赖性地促进ANGPTL3 mRNA和蛋白质表达;AN-GPTL3的mRNA和蛋白质表达量还受环境胆固醇浓度影响.ANGPTL3的表达可能与糖尿病、高胆固醇血症密切相关.     

丙型肝炎病毒阳性血清体外感染人肝细胞的研究

目的 研究丙型肝炎病毒(HCV)抗体(Ab)阴性,HCV-RNA阳性血清建立体外感染肝细胞模型.方法 HCV Ab阴性,HCV-RNA阳性的窗口期血清与人肝细胞共同培养,用反转录-聚合酶链反应(RT-PCR)、免疫荧光染色、Western blot、共聚焦显微镜和透射电镜等方法检测细胞内HCV核酸复制、蛋白质表达及超微结构改变.结果 细胞与病毒共同培养7～45 d,细胞内和/或培养上清中可间断检出HCV正、负链RNA;细胞浆内有HCV 核心和NS3抗原的表达;细胞超微结构有改变,并于感染后第24天时观察到类似病毒样颗粒.结论 窗口期血清中的HCV能在人肝细胞7701中复制一段时间.     

Enhancement of natural killer cell activity in human immunodeficiency virus-infected subjects by in vitro treatment with biologic response modifier OK-432.

A decrease in natural killer (NK) cell function has been related to the progression of human immunodeficiency virus (HIV) infection. In the present study, we assessed the ability of a streptococcus-derived biologic response modifier, OK-432, to augment NK lysis of uninfected K562 and U937 cells and HIV-infected U937 cells by peripheral blood mononuclear cells (PBMC) from HIV-seropositive homosexual men. Optimal two- to fourfold increases in lysis of the three targets were observed after pretreatment of PBMC from HIV-negative subjects for 4 h with 2 micrograms of OK-432 per ml. This effect was related primarily to gamma interferon (IFN-gamma) production induced by OK-432 and was not linked to production of tumor necrosis factors alpha and beta or to monocytes in the cultures. The enhancing effect of OK-432 on NK cell function was diminished but still evident in PBMC from subjects with relatively early-phase (< 3-year) HIV infection and high CD4+ cell counts and was lower in subjects with longer-term HIV infection (> 3 years), in association with reduced production of IFN-gamma. Augmentation of NK cell activity in HIV-infected men by OK-432 was comparable to that induced by treatment of cells with 1,000 U of IFN-alpha or interleukin 2 per ml. The data suggest that the NK cell-enhancing effects of OK-432 are at least in part mediated by IFN-gamma and that OK-432 may be effective in treatment of patients with early-phase HIV infection.