Real-time quantification of hepatitis B virus core-promoter and pre-core mutants during hepatitis E antigen seroconversion

BACKGROUND/AIMS: Detection of hepatitis B virus (HBV) core-promoter A(1762)T-G(1764)A and pre-core G(1896)A mutants has relied on qualitative assays. We tested the hypothesis that the quantity of A(1762)T-G(1764)A and G(1896)A mutants might have clinical impact, by quantifying these mutants before and after HBe antigen (HBeAg) seroconversion in 58 patients. METHODS: A real-time quantitative-polymerase chain reaction (Q-PCR) was developed, using minor groove binder (MGB)-conjugated TaqMan probes to impart reaction specificity for wildtype/mutant HBV populations. RESULTS: Significant quantities (>20%) of core-promoter A(1762)T-G(1764)A mutant existed in 65% of patients before and after HBeAg seroconversion, and were significantly changed (>20% increase/decrease) in 13% of patients after seroconversion. Quantity of A(1762)T-G(1764)A mutants was positively correlated with alanine aminotransferase (ALT) (P<0.001) and HBV DNA (P<0.001) levels, both before and after HBeAg seroconversion. Significant quantities of pre-core G(1896)A mutant existed in about 90% of patients before and after HBeAg seroconversion, and were changed in 16% of patients after seroconversion. Quantity of G(1896)A mutant was negatively correlated with ALT (P=0.044) and HBV DNA (P=0.007) levels. CONCLUSIONS: The A(1762)T-G(1764)A and G(1896)A mutants existed in a high proportion of patients before and were unaffected after HbeAg seroconversion. The quantities of A(1762)T-G(1764)A mutant were positively and G(1896)A mutant negatively correlated with liver inflammation and viral replication.     

Vaccine and skin testing properties of two avirulent Mycobacterium bovis mutants with and without an additional esat-6 mutation

SETTING: Molecular techniques are now available to develop new live tuberculosis vaccines by producing avirulent strains of the Mycobacterium tuberculosis complex with known genes deleted. OBJECTIVES: Determine if removal of esat-6 from new live tuberculosis vaccines with known attenuating mutations affects their vaccine efficacy and if it could enable the development of discriminating diagnostic tests. DESIGN: Remove the esat-6 gene by allelic exchange from two illegitimate mutants of Mycobacterium bovis that had previously been shown to have similar vaccine efficacy to BCG in a guinea pig vaccination model. Determine the effect this removal has on virulence, vaccine efficacy and skin test reactivity in guinea pigs. RESULTS: Two double knockout strains of M. bovis were produced and their virulence and vaccine efficacy were compared to their parent strains. Removal of the esat-6 gene had no significant effect on vaccine efficacy. In skin tests, animals inoculated with the double knockout strains reacted to PPD but not ESAT-6, whereas those inoculated with the parent strains had similar skin test reactivity to both PPD and esat-6. CONCLUSION: Removal of esat-6 from new live tuberculosis vaccine candidates has no significant effect on vaccine properties but does enable the use of skin tests to distinguish between vaccination and infection.     

Pathogenesis of hepatitis B virus infection

Infection with hepatitis B virus (HBV) leads to a wide spectrum of clinical presentations ranging from an asymptomatic carrier state to self-limited acute or fulminant hepatitis to chronic hepatitis with progression to cirrhosis and hepatocellular carcinoma. Infection with HBV is one of the most common viral diseases affecting man. Both viral factors as well as the host immune response have been implicated in the pathogenesis and clinical outcome of HBV infection. In this review, we will discuss the impact of virus-host interactions for the pathogenesis of HBV infection and liver disease. These interactions include the relevance of naturally occurring viral variants for clinical disease, the role of virus-induced apoptosis for HBV-induced liver cell injury and the impact of antiviral immune responses for outcome of infection.     

拉米夫定与HBV YMDD耐药变异及临床相关因素分析

目的:用PCR-RFLP方法检测慢性乙型肝炎患者HBV-YMDD变异发生的情况,并分析与YMDD变异发生有关的因素。方法:从慢性乙型肝炎患者血清中提取的HBV DNA,扩增HBV多聚酶YMDD主型区核苷酸序列,用针对突变位点的特异限制性内切酶酶切扩增产物,经6％聚丙烯酰胺凝胶电泳后用限制性片段长度多态性技术鉴定HBVYMDD变异。结果:在152例慢性乙型肝炎患者中,72例单用拉米夫定治疗、41例接受拉米夫定与干扰素联合治疗、39例未接受拉米夫定治疗的患者,HBV YMDD变异检出率分别是47.2％、19.5％、7.7％;72例单用拉米夫定治疗的患者,36例治疗2周～6个月、20例治疗7～12个月、16例治疗13～27个月,HBV YMDD变异检出率分别是25％、55％、87.5％。结论:在使用拉米夫定治疗慢性乙型肝炎的过程中可发生YMDD耐药变异,且随拉米夫定治疗时间的延长,变异发生率增加,但拉米夫定与干扰素联合进行抗病毒治疗可延迟或阻滞YMDD变异的发生。HBVYMDD变异可能自然存在或发生。HBV基因组其他位点的变异也可能使HBV对拉米夫定产生耐药性。     

Gene expression in brain: a window on ethanol dependence,neuroadaptation, and preference

This article represents the proceedings of a symposium at the 2002 joint RSA/ISBRA Conference in San Francisco, California. The organizer was Paula L. Hoffman and the co-chairs were Paula L. Hoffman and Michael Miles. The presentations were (1) Introduction and overview of the use of DNA microarrays, by Michael Miles; (2) DNA microarray analysis of gene expression in brains of P and NP rats, by Howard J. Edenberg; (3) Gene expression patterns in brain regions of AA and ANA rats, by Wolfgang Sommer; (4) Patterns of gene expression in brains of selected lines of mice that differ in ethanol tolerance, by Boris Tabakoff; (5) Gene expression profiling related to initial sensitivity and tolerance in gamma-protein kinase C mutants, by Jeanne Wehner; and (6) Gene expression patterns in human alcoholic brain: from microarrays to protein profiles, by Joanne Lewohl.     

FK506-binding protein of Neurospora crassa (NcFKBP) mediates sensitivity to the immunosuppressant FK506; resistant mutants identify two loci

Summary Growth of Neurospora crassa wild-type is inhibited by micromolar concentrations of the immuno-suppressive macrolide FK506. Spontaneous and induced mutations that confer resistance to FK506 identified two loci, fkr-1 and fkr-2. They map on the right arm of linkage group V on either side of inl with fkr-1 being centromere proximal. Allele fb (fkr-2) lacks immunodetectable N. crassa FK506-binding protein (NcFKBP). This demonstrates that the sensitivity of N. crassa towards FK506 is mediated by NcFKBP. FK506-binding proteins have been shown to be highly conserved, i.e., found in all eukaryotic cells tested, and to exhibit peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro. Possible functions for the loci are discussed. Apart from the resistance to FK506 no other mutant phenotype was detected not even in double mutants that lacked NcFKBP as well as cyclophilin. Cyclophilin mediates the cytotoxic effect of the immunosuppressive drug Cyclosporin A and is also characterized by PPIase activity in vitro. Both FK506-resistant alleles studied exhibit incomplete dominance in forced heterokaryons. A mechanism is proposed to explain this dominance especially in view of the NcFKBP-deficient allele, fb.     

Site-directed mutagenesis provides insights into the selective binding of trityl derivatives to Plasmodium falciparum dUTPase

We have previously identified a series of triphenylmethane derivatives of deoxyuridine with antimalarial activity in vitro which selectively inhibit Plasmodium falciparum deoxyuridine triphosphate nucleotidohydrolase (PfdUTPase) compared to the human enzyme. The crystal structure of PfdUTPase in complex with one of these inhibitors suggested that the triphenylmethane derivative was selective due to a series of interactions between the trityl group and the side chains of residues Phe⁴⁶, Ile¹¹⁷ and Lys⁹⁶ located in a hydrophobic pocket distinct from the phosphate binding site. Here we show by site-directed mutagenesis that the hydrophobic nature of the trityl binding site and in particular aromatic interactions established between the inhibitor and residue Phe⁴⁶ contribute significantly to the binding of uracil-based derivatives containing trityl groups in the 5′-position. Thus, changing Phe⁴⁶ for alanine resulted in increased K_i values for all compounds tested. Conversely, substitution of the polar residue Lys⁹⁶ for Ala results in smaller K_i values and an increase in selectivity with regard to human dUTPase. This information will aid in the design of inhibitors with improved activity against the Plasmodium enzyme.     

Muscle mitochondrial DNA in encephalomyopathy and ragged red fibres: a Southern blot analysis and literature review

Summary Various mitochondrial DNA abnormalities have been described in patients with encephalomyopathies. We performed Southern blot analysis of skeletal muscle mitochondrial DNA in nine adult patients with clinical features and ragged red fibres suggesting mitochondrial dysfunction. Two patients with encephalomyopathy and two with the MERRF syndrome (myoclonus epilepsy with ragged red fibres) had the normal PvuII restriction pattern of muscle mitchondrial DNA. In contrast, mitochondrial DNA deletion was observed in two of six patients with ophthalmoplegia. One suffered from typical Kearns-Sayre syndrome and the other from isolated external ophthalmoplegia. None of these patients had affected relatives. The detection of mitochondrial DNA deletion in external ophthalmoplegia and their site and size support previously reported data.     

重组pEGFP-C2-p100-TSN点突变质粒构建及表达

目的 将6个不同的p100-TSN.Mutants基因片段分别定向连入PEGFP-C2质粒中,使P100-TSN突变蛋白能够与绿色荧光蛋白在COS7细胞中融合表达,从而为进一步研究p100蛋白TSN结构域的功能奠定实验基础. 方法 利用EcoR Ⅰ和XhoⅠ双酶切方法从6个pcDNA3.1 (+) -p100-TSN.Mutants重组质粒中分别获得p100-TSN.Mutants的cDNA片段,将其连入pEGFP-C2质粒载体中,再将成功构建的6个pEGFP-C2-p100-TSN.Mutants质粒分别转染COS7细胞中,荧光显微镜下观察绿色荧光蛋白表达.结果 ①将重组质粒进行双酶切鉴定可见p100-TSN.Mutants的cDNA片段;②转染重组质粒后可观察到绿色荧光蛋白的表达.结论 ① 6个pEGFP-C2-p100-TSN.Mutants重组质粒构建成功;② p100-TSN突变蛋白可与绿色荧光蛋白在COS7细胞中融合表达.