Biological impacts of“hot-spot” mutations of hepatitis B virus X proteins are genotype B and C differentiated

AIM: To investigate the biological impacts of "hot-spot" mutations on genotype B and C HBV X proteins (HBx). METHODS: Five types of "hot-spot" mutations of genotype B or C HBV X genes, which sequentially lead to the amino acid substitutions of HBx as I127T, F132Y, K130M+V131I, I127T+K130M+V131I, or K130M+V131I+F132Y, respectively, were generated by means of site-directed mutagenesis. To evaluate the anti-proliferative effects, HBx or related mutants' expression vectors were transfected separately to the Chang cells by lipofectamine, and the cells were cultured in hygromycin selective medium for 14 d, drug-resistant colonies were fixed with cold methanol, stained with Giemsa dyes and scored (increase of the colonies indicated the reduction of the anti-proliferation activity, and vice versa). Different types of HBx expression vectors were co-transfected separately with the reporter plasmid pCMVβ to Chang cells, which were Iysed 48 h post-transfection and the intra-cellular β-galactosidase activities were monitored (increase of the β-galactosidase activities indicated the reduction of the transactivation activity, and vice versa). All data obtained were calculated by paired-samples t-test. RESULTS: As compared to standard genotype B HBx, mutants of I127T and I127T+K130M+V131I showed higher transactivation and anti-proliferative activities, while the mutants of F132Y, K130M+V131I, and K130M+V131I+F132Y showed lower activities. As compared to standard genotype C HBx, I127T mutant showed higher transactivation activity, while the other four types of mutants showed no differences. With regard to anti-proliferative activity, compared to standard genotype C HBx, F132Y and K130M+ V131I mutants showed lower activities, and K130M+V131I +F132Y mutant, on the other hand, showed higher activity, while the mutants of I127T and I127T+K130M+V131I showed no differences, CONCLUSION: "Hot-spot" mutations affect the anti-proliferation and transactivation activities of genotype B and/or C HBx, and the biological impacts of most "hot-spot" mutations on HBx are genotype B and C differentiated.     

晚期糖基化终末产物对内皮细胞前列腺素合成的影响及可能机制

目的探讨晚期糖基化终末产物对内皮细胞前列环素、前列腺素E2合成的影响及可能机制。方法不同浓度的晚期糖基化终末产物作用内皮细胞,酶联免疫吸附法测定前列环素的稳定代谢产物6-酮—前列腺素F1α和前列腺素E2的表达。逆转录聚合酶链反应、免疫细胞化学测定环氧合酶2 mRNA和蛋白表达的改变。晚期糖基化终末产物受体、核因子κB单/双基因反义RNA对晚期糖基化终末产物刺激内皮细胞分泌6-酮—前列腺素F1α、前列腺素E2的影响。结果晚期糖基化终末产物引起内皮细胞分泌6-酮—前列腺素F1α、前列腺素E2增加(P<0.01),具有剂量依赖关系。晚期糖基化终末产物引起内皮细胞环氧合酶2的mRNA和蛋白表达增加(P<0.01)。晚期糖基化终末产物受体、核因子κB单/双基因反义RNA可减轻晚期糖基化终末产物刺激的内皮细胞分泌6-酮—前列腺素F1α、前列腺素E2(P<0.05或0.01),双基因的抑制效果更明显(P<0.05)。结论晚期糖基化终末产物可能通过晚期糖基化终末产物受体、核因子κB途径使内皮细胞的环氧合酶2表达增加,从而引起前列环素、前列腺素E2合成增加,这个机制可能参与了晚期糖基化终末产物所致的血管炎症损伤。     

弓形虫表面抗原P22蛋白的研究进展

*　弓形虫(Toxoplasma gondii)是专性寄生于人和多种动物组织有核细胞内的原虫,呈世界性分布。近年来关于弓形虫细胞表面抗原作为诊断试剂及免疫疫苗的双重潜在价值得到广泛研究,主要发现有5种表面抗原:P22、P23、P30、P35、P43。其中P22基因依据克隆的顺序也被称为SAG2。本文就弓形虫重要抗原P22的基因和蛋白结构,及该抗原在弓形虫虫株的分型、血清诊断、疫苗方面的研究作简要介绍。1　关于弓形虫P22基因弓形虫的生活史中除了合子期外,其他期的核均为单倍体。已经确定的染色体有8条, 各编码染色体分子质量单位分别为: 2.0、2.4、2.6、…     

胸腺细胞凋亡的死亡信号表达

目的:探讨胸腺细胞凋亡过程中死亡信号通路的存在依据及其意义。方法:选用2月龄清洁级SD大鼠,用ABC法检测胸腺细胞Fas/FasL、Caspase-3、Caspase-8、Caspase-12表达,用RT-PCR法检测培养胸腺细胞的TNF-β和Caspase-3表达。结果:Fas在胸腺皮、髓质细胞弥散均匀分布,FasL阳性细胞较少,Caspase-3主要分布于皮质胸腺细胞,Caspase-8则分布于胸腺髓质,Caspase-12在皮、髓质胸腺细胞均匀分布,RT-PCR检测显示胸腺细胞均表达TNF-β和Caspase-3。结论:大鼠胸腺内存在完整的细胞凋亡死亡信号通路的各种成分。死亡信号通路在胸腺细胞凋亡过程中可能扮演重要角色。     

An immuno-dot blot assay for the detection of antibody to HIV

This paper describes a simple and rapid immuno-dot blot assay for the detection of antibody to HIV. It utilizes nanogram quantities of HIV lysate, immobilized on nitrocellulose paper, and microliter quantities of serum or culture supernatants for the detection of antibody. The test is highly sensitive and specific. It offers the opportunity to perform multiple assays at a minimal cost and provides the additional advantage of not requiring any sophisticated or expensive equipment to perform the test or interpret the results.     

大鼠骨髓基质细胞体外诱导分化和脑内定位移植

目的 了解骨髓基质细胞（MSC）在体外对抗氧化剂和生长因子等的反应及其脑内移植后的情况。方法 体外培养成年大鼠MsC，用β-巯基乙醇、BDNF、GDNF、CNTF、forskolin诱导分化，并将MSC植入成鼠大脑皮质，观察MSC在体内外环境中是否能够分化为神经细胞。采用流式细胞术鉴定MSC，免疫荧光染色法鉴定神经细胞。结果 在本实验条件下，MSC未见被诱导成神经细胞，植入脑内的MSC能够存活并迁移，但也未见分化为神经细胞。结论 MSC横向分化为神经细胞可能是多因素作用的结果，需要更多的实验对其横向分化和定向分化进行探索。     

Continuous regional arterial infusion and laparotomic decompression for severe acute pancreatitis with abdominal compartment syndrome

AIM:To evaluate the therapeutic effects of abdominal decompression plus continuous regional arterial infusion(CRAI) via a drug delivery system(DDS) in severe acute pancreatitis(SAP) patients with abdominal compartment syndrome(ACS).METHODS:We presented our recent experience in 8 patients with SAP.The patients developed clinical ACS,which required abdominal decompression.During the operation,a DDS was inserted into the peripancreatic artery(the catheter was inserted from the right gastroepiploic artery until...     

Hepatitis B spliced protein (HBSP) generated by a spliced hepatitis B virus RNA participates in abnormality of fibrin formation and functions by binding to fibrinogen γ chain

Hepatitis B spliced protein (HBSP) encoded by a 2.2 kb singly spliced hepatitis B virus (HBV) pre-genomic RNA (spliced between positions 2447 and 489 nt) is involved in the pathogenesis of HBV infection, whereas the exact mechanism is far from being fully elucidated. In this study, a yeast two-hybrid system using HBSP as bait was employed to screen binding partners for HBSP from a human liver cDNA library. The interaction between HBSP and fibrinogen γ chain (FGG) was further confirmed in vitro using a GST pull-down assay and confirmed in vivo using a mammalian two-hybrid assay and co-immunoprecipitation. It was identified that this interaction is mediated by the N terminal 47 amino acid residues of HBSP. HBSP could inhibit fibrin polymerization, factor XIIIa-mediated fibrin cross-linking, adhesion of platelets to fibrinogen and ADP-stimulated platelet aggregation. However, the interaction-mediating fragment 1-47 of HBSP is not sufficient for the inhibitory activity on fibrinogen function. The findings suggested that HBSP may participate in the hemostatic abnormality in patients with HBV-related liver diseases.     

Influence of enterococci on human sperm membrane in vitro

Aim： To study the influence of enterococci on human sperm membrane in vitro. Methods： Ejaculated human sperm were artificially infected with β-hemolytic or non-β-hemolytic enterococci at the bacteria： sperm ratio of 50：1 at 37℃. Sperm membrane integrity was examined after incubation for 1, 3 and 5 h by hypoosmotic swelling （HOS） test and electron microscopy. Results： Sperm infected with β-hemolytic enterococci had lower HOS scores compared with non-β-hemolytic strains or uninfected control （P 〈 0.01）. The HOS test scores of sperm infected with β-hemolytic enterococci increased in the presence of phosphatidylcholine, an inhibitor of hemolysin. Non-β-hemolytic strains showed no significant difference in swelling rate, compared with the control group （P 〉 0.05）. It was shown by electron microscopy that β-hemolytic enterococci caused significant rupture of human sperm membrane. Conclusion： β-hemolytic enterococci caused human sperm membrane injury, and might be mediated by the hemolysin of enterococci.