C基因截短的HBV复制与包装

目的 探讨C基因截短型HBV变异体的复制与包装。方法 采用分子克隆、人工定点突变等技术构建C基因截短型HBV变异体质粒，用脂质体法转染HepG2细胞，提取细胞内及培养上清液中DNA分别进行Southem杂交，PCR及实时定量荧光PCR分析。结果 经DNA测序及酶切鉴定证实C基因截短型HBV质粒载体构建成功；C基因截短型HBV为复制缺损型，与辅助质粒共转染HepG2细胞，可在细胞内及培养上清液中检测到HBV各种DNA构型；DNA定量分析提示C基因截短型HBV的包装效率较野生型HBV提高3～40倍。结论 C基因截短型HBV变异体为复制缺损型，单独转染后不能在肝细胞内包装与复制，但在缺失包装信号ε的相应辅助病毒辅助下可有效复制并包装成子代病毒颗粒分泌到胞外，且包装效率大大提高。     

Postoperative spindle cell nodule of the breast: Pseudosarcomatous myofibroblastic proliferation following endo‐surgery

Despite the frequent use of fine‐needle aspiration, core biopsy and surgery, postoperative spindle cell nodule (PSCN) is a rare pathological complication that may be diagnostically treacherous. Presented herein is the case of a 52‐year‐old woman who developed a 7 mm mammary nodular lesion 66 days after removal of an area of columnar cell hyperplasia involving cellular and architectural atypia, performed with the Mammotome Breast Biopsy System. The lesion was highly cellular and composed of intersecting fascicles of plump spindle cells with blunt‐ended elongated nuclei and nucleoli easily visible. Interspersed mononuclear cells and hemosiderin‐laden macrophages were evident. PSCN is a reactive, benign myofibroblastic proliferation. Differential diagnosis includes benign and malignant spindle cell lesions of the breast. Recognition of this reactive lesion will avoid overdiagnosis of spindle cell malignant tumor. Attention to clinicopathological and histological features should result in accurate recognition of this lesion.     

血管紧张素原基因5′端核心启动子区(-6)A-G和(-20)A-C变异与哈萨克族人原发性高血压相关性分析

目的　研究血管紧张素源 (angiotensinogen,AGT)基因核心启动子区域 (- 6 ) A- G和 (- 2 0 )A- C位点变异与哈萨克族人原发性高血压相关关系。方法　采用经典的饱和酚 /氯仿抽提法提取哈萨克族正常人 74名和高血压患者 12 5例白细胞基因组 DNA,通过 PCR、单链构象多态性、限制性片段长度多态性和测序等技术 ,鉴定不同个体 AGT基因核心启动子区域 (- 6 )、(- 2 0 )位等位基因的类型 ,观察在高血压组和正常血压组不同基因型的分布和等位基因频率的差异。结果　 (1)哈萨克族人 AGT基因 - 16 4～ 73区域仅存在 (- 6 ) A- G、(- 2 0 ) A- C两种变异。(2 ) AGT基因 (- 6 )位点 AA、AG、GG基因型的频率在高血压组和正常血压组分别为 0 .39、0 .4 5、0 .16和 0 .4 9、0 .4 9、0 .0 2 ,两组之间差异存在显著性 (χ2 =8.5 6 ,P=0 .0 14 )。A、G等位基因频率分别为 0 .6 2、0 .38和 0 .73、0 .2 7,差异存在显著性 (χ2 =5 .35 ,P=0 .0 2 1)。(3) AGT基因 (- 2 0 )位点 AA、AC、CC基因型频率在高血压组和正常血压组分别为 0 .6 9、0 .2 6、0 .0 5和 0 .6 5、0 .32、0 .0 3,差异无显著性 (χ2 =2 .4 2 ,P=0 .30 ) ;A、C等位基因的频率分别为 0 .82、0 .18和 0 .82、0 .18,差异无显著性 (χ2 =0 ,P=0 .99)。(4) AGT基     

Probable implication of mutations of the X open reading frame in the onset of fulminant hepatitis B

A pathogenic role of precore-defective mutation in the onset of fulminant hepatitis B has been suggested. However, precore-defective mutants do not always cause fulminant hepatitis B and are not always isolated from affected patients. These findings strongly suggest the presence of some additional important mutations outside the precore region in fulminant hepatitis. In the present investigation an attempt was made to sequence the X open reading frame of hepatitis B virus DNA isolated from seven patients with fulminant hepatitis B and five patients with acute hepatitis B. The latter were used as controls. Since the X open reading frame encodes the X protein and contains the core promoter/enhancer II complex, some critical mutations may enhance or disrupt the replication and expression of hepatitis B virus DNA leading to fulminant hepatitis. A C-to-T substitution was found at nucleotide (nt) 1655, an A-to-T substitution at nt 1764 and a G-to-A substitution at nt 1766 in 4, 5 and 5 patients, respectively, out of the seven with fulminant hepatitis. These substitutions were not recognized in the patients with acute hepatitis. These mutations might change the function of the X protein and core promoter/enhancer II complex. It is suggested, therefore, that these mutations, as well as the precore-defective mutation, may play an important role in the pathogenesis of fulminant hepatitis. © Wiley-Liss, Inc.     

Changes in hypothalamic temperature of rats after daily exposure to heat at a fixed time

Rats were subjected to an ambient temperature (T_a) of 33°C for ca. 5 h during the last half of the dark phase for 5, 14 or 28 consecutive days (heat-exposed rats, HE), while control rats were kept at a constant T_a of 24°C. After the heat exposure schedule, the levels of hypothalamic temperature (T_hy) as an index of body core temperature in the HE were significantly lower than those of the controls for 2–4 h in the last half of the dark phase. The low levels of T_hy persisted during the specific period for 1, 3 and 6 days after the end of the 5-, 14- and 28-day heat exposure schedules, respectively. These results confirm that, in rats subjected to daily heat exposure for ca. 5 h at a fixed time per day, their T_hy falls during the period when the rats were previously exposed to heat, and suggest that the duration of the specific T_hy change observed after completing the heat exposure schedule depends on the length of the heat exposure schedule.     

丙型肝炎病毒核心蛋白在HepG2细胞中的表达及其对端粒酶活性的影响

目的 建立HCV核心蛋白细胞表达模型，并探讨其对细胞端粒酶活性的影响。方法 用PCR法扩增出HCV核心基因cDNA，将其插入真核表达载体pBK-CMV的HindⅢ和BamHⅠ位点间，构建重组质粒pBK-HCVc。再将重组质粒pBK-HCVc和空载体分别导入肝癌细胞株HepG2中，G418筛选，RT－PCR、免疫组化和蛋白印迹鉴定HCV核心蛋白表达。PCR－ELISA法检测端粒酶活性。结果 构建的pBK-HCVc质粒在HepG2细胞中有稳定表达。表达HCV核心蛋白的细胞HepG2-C的端粒酶活性较转染空载体的细胞HepG2-CMV明显升高。结论 HCV核心蛋白上调了端粒酶活性，可能是HCV诱发肝细胞癌的一种途径。     

Molecular and serological evaluation of surface antigen negative hepatitis B virus infection in blood donors from Venezuela

Surface antigen negative hepatitis B virus (HBV) infection was evaluated in Venezuela, by molecular characterization of blood samples positive for antibodies to core antigen (anti-HBc) and negative for surface antigen (HBsAg) in blood donors (residual infections). HBV DNA was found in 11/258 samples (4.3%), and was significantly associated with high levels of anti-HBc antibodies (>25 UI/ml, P < 0.05), while no correlation was found between the presence of HBV DNA and the levels of anti-HBs. Synonymous and non-synonymous mutations were found in the HBV surface region (but not vaccine escape mutants) and in the precore/core region (precore mutants in 2/7 samples and 33-45 bp deletions near the N-terminal core region in 4/19 samples). While HBV genotype F prevails among HBsAg positive samples from blood donors in Venezuela, residual infection isolates were mainly genotypes A and D. Phylogenetic analysis of viral surface and core region revealed discrepancies in genotype designation in 6/9 samples, suggesting the presence of mixed infection or recombination. In conclusion, HBV residual infection in Venezuela does not seem to be frequently observed in HBV genotype F. This type of infection is frequently associated with variants exhibiting mutations in the surface gene that might be affecting the correct recognition by commercial tests, with precore mutants and with core internal deletions. These variants do not seem to cause severe liver disease, and on the contrary, were found circulating at low viremia.     

Transcription from the gene encoding the herpesvirus entry receptor nectin-1 (HveC) in nervous tissue of adult mouse

Hepatitis C virus core protein, in addition to being a component of the viral capsid, has a number of regulatory functions. Here we showed two bodies of evidence indicating that a fraction of the core protein species is a substrate of the ubiquitin (Ub)-proteasome pathway of targeted proteolysis. First, the core protein processing the C-terminal hydrophobic region is metabolically unstable, and incubation with a proteasome inhibitor led to a significant accumulation of the protein. Second, an in vivo ubiquitylation assay indicates conjugation of multi-Ub chain to the unstable core protein. In contrast, a stable form of core protein, p21, is also able to be ubiquitylated, but it links to a single or only a few Ub moiety. Therefore, processing event(s) at the C-terminal hydrophobic domain of HCV core protein may affect the ubiquitylation pathway, particularly the efficiency of the multi-Ub chain assembly, resulting in stable, matured core proteins.     

蛋白激酶R与丙型肝炎病毒核心蛋白相互作用区域定位

目的： 观察丙型肝炎病毒（HCV）核心蛋白（CP）对蛋白激酶R(PKR)表达的影响；定位PKR与CP直接结合的区域。方法: 对Huh-7、转染表达CP的Huh-7及含有全长HCV复制子(replicon) Huh-7细胞株的PKR表达水平及干扰素（IFN）诱导前后replicon Huh-7细胞中HCV结构蛋白和非结构蛋白表达水平作比较；对CP与PKR进行免疫共沉淀试验、谷胱苷肽S转移酶（GST）结合试验。结果: Replicon Huh-7中PKR表达水平高于Huh-7及转染表达CP的Huh-7；IFN诱导后PKR表达增加，且明显抑制HCV结构和非结构蛋白的表达；PKR能与CP直接结合，依赖于PKR的N端1-180氨基酸（aa）。结论: CP能直接作用于PKR N端1-180 aa，导致PKR组成性激活，从而干扰PKR介导的相关信号转导通路。CP与PKR的相互作用是HCV病毒蛋白与细胞蛋白相互作用又一新的模式，在HCV持续感染及肝癌2者发病机制方面可能起重要作用。     

Cardiac ankyrin repeat protein is preferentially induced in atrophic myofibers of congenital myopathy and spinal muscular atrophy

Cardiac ankyrin repeat protein (CARP), which is structurally characterized by the presence of four ankyrin repeat motifs in its central region, is believed to be localized in the nucleus and to participate in the regulation of cardiac-specific gene expression in cardiomyocytes. However, we recently found that CARP was induced in skeletal muscle by denervation, leading us to speculate that CARP may be induced under some pathological conditions. In the present study, we immunohistochemically analyzed the expression of CARP in 11 cases of spinal muscular atrophy (SMA) and 14 cases of congenital myopathy. In SMA, CARP was expressed selectively in severely atrophic myofibers, suggesting that CARP expression may reflect the status of muscle atrophy. Furthermore, in the congenital myopathies, the expression patterns of CARP were distinct among the subtypes, which included nemaline myopathy, myotubular myopathy, central core disease, and congenital fiber type disproportion. Although CARP was preferentially expressed in severely damaged myofibers in nemaline myopathy, it was not detected in central core disease. These findings suggest that immunohistochemical evaluation of CARP may be helpful in the diagnosis of SMA and the congenital myopathies.