HBV－NC1株X基因在真核细胞中表达，基因调控与致癌作用的初探

报道了ＨＢＶ－ＮＣ１株的Ｘ基因进入真核细胞表达载体ｐＸＴ１质粒的ＴＫ启动子之后,转染细胞及表达Ｘ基因成功。即发现包装病毒感染ＮＩＨ／３Ｔ３细胞后有Ｘ基因表达,用斑点杂交又证明此Ｘ基因已整合到细胞的基因组中,当与有ｐＭＳＨ报告基因的质粒共同感染真核细胞后确有激活转录调控现象出现。应用反义的ＸＲＮＡ表达载体感染肝癌细胞发现有接触抑制作用出现。     

Agrin-Deficient Myotube Retains Its Acetylcholine Receptor Aggregation Ability when Challenged with Agrin

Abstract: Agrin is a synapse-organizing molecule that mediates the nerve-induced aggregation of acetylcholine receptors (AChRs) and other postsynaptic components at the developing and regenerating vertebrate neuromuscular junctions. At the neuromuscular junction, three different cell types can express agrin, i.e., neuron, muscle, and Schwann cell. Several lines of evidence suggested that neuron-derived agrin is the AChR-aggregating factor, but the possible roles of muscle-derived agrin in the formation of AChR aggregate are not known. By using the recombinant DNA method, a clonal stable C2C12 cell line transfected with antisense agrin cDNA was created. RNA dot blot and western blot analysis indicated that the expression of agrin in the transfected cell was abolished by DNA transfection. When the agrin-deficient C2C12 cells were induced to form myotubes and subsequently cocultured with agrin cDNA transfected fibroblasts, AChR aggregates were formed in the cocultures. In addition, acetylcholinesterase (AChE) aggregates in agrin-deficient myotubes were also induced by exogenous agrin and the AChE aggregates were colocalized with the AChR aggregates. The agrin-deficient myotubes could also respond to neuron-induced AChR aggregation after coculturing with neuroblastoma cells. Thus, the agrin-deficient myotubes retain their ability to exhibit the agrin- or neuron-induced AChR aggregation. This result suggests that the formation of postsynaptic specializations during development and regeneration is mediated by neuron-derived agrin but not the agrin from muscle.     

Design and synthesis of chiral peptidic nucleic acids

Summary Due to the increasing interest in the use of oligonucleotide analogues as antisense and antigene drugs, we designed a chiral analogue constituted of a peptidic frame bearing nucleobases in suitable positions (C-PNA). We recently reported the synthesis of four nonnatural α-amino acids with the DNA bases in the lateral chain. In this paper we present an improved synthesis of the Fmoc monomers and their polymerisation to polypeptidic oligonucleotide analogues using a modification of the standard protocol for solid phase peptide synthesis.     

CNS Drug Design Based on Principles of Blood-Brain Barrier Transport

Abstract: Lipid-soluble small molecules with a molecular mass under a 400–600-Da threshold are transported readily through the blood-brain barrier in vivo owing to lipid-mediated transport. However, other small molecules lacking these particular molecular properties, antisense drugs, and peptide-based pharmaceuticals generally undergo negligible transport through the blood-brain barrier in pharmacologically significant amounts. Therefore, if present day CNS drug discovery programs are to avoid termination caused by negligible blood-brain barrier transport, it is important to merge CNS drug discovery and CNS drug delivery as early as possible in the overall CNS drug development process. Strategies for special formulation that enable drug transport through the blood-brain barrier arise from knowledge of the molecular and cellular biology of blood-brain barrier transport processes.     

Normal starch content and composition in tubers of antisense potato plants lacking D-enzyme (4-α-glucanotransferase)

Transgenic potato (Solanum tuberosum L.) plants were created with sense and antisense copies of the potato D-enzyme (disproportionating enzyme; EC␣2.4.1.25) cDNA linked to patatin and cauliflower mosaic virus 35 S promoters, and screened for D-enzyme activity in tubers. Transformants with sense constructs mostly had wild type D-enzyme activity but two plants had only about 1% wild-type activity. Transformants with antisense constructs had activity ranging from 90% to about 1% of wild type. Three 35 S antisense plants with very low activity were analysed in detail. Western blot analysis showed that D-enzyme was present in greatly reduced amounts in tubers and in leaves, whereas plastidic starch phosphorylase (EC 2.4.1.1) was unaffected. The lack of D-enzyme resulted in slow plant growth but development was otherwise apparently normal. Furthermore, the starch content of tubers was not appreciably altered in amount, proportion of amylose, molecular weight of debranched amylopectin, or branch chain length, despite the lack of D-enzyme. These results do not indicate a direct requirement for D-enzyme in the synthesis and accumulation of storage starch in tubers. The results are discussed in terms of the known reactions catalysed by D-enzyme and possible involvement of D-enzyme in starch metabolism. Received: 12 November 1997 / Accepted: 23 December 1997     

Circular Dichroism Studies of the Interaction of Tat Analogues Substituted in the Arg52 Position with TAR RNA HIV-1

The Tat wild-type fragment of sequence Arg⁴⁹-Lys-Lys-Arg⁵²-Arg-Gln-Arg-Arg-Arg⁵⁷-NH₂ (labelled as Tat1) and three analogues of this fragment with the substitution Arg⁵² D-Arg⁵² (labelled as Tat2) or L-citrulline (Cit) (labelled as Tat3) or L-ornithine (Orn) (labelled as Tat4) were synthesized to study Tat-TAR RNA HIV-1 (27-nucleotide fragment of sequence 5-AGAUCUGAGCCUGGAGCUCUCU-3) interactions by circular dichroism. -helical structure was the most readily adopted by the Tat3 analogue with Arg⁵² Cit substitution. All the peptides investigated caused conformational changes in the TAR structure. The most dramatic changes were observed for the Tat2-TAR complex.     

反义RNA调节肿瘤细胞O~6-甲基鸟嘌呤-DNA甲基转移酶活性

报道了逆转录病毒载体介导的反义ＲＮＡ对肿瘤细胞Ｏ６－甲基鸟嘌呤－ＤＮＡ甲基转移酶（ＭＧＭＴ）活性的调节作用．构建了三个表达ＭＧＭＴ反义ＲＮＡ的逆转录病毒载体并用它们转染ＨｅＬａＳ３肿瘤细胞,观察细胞在转染前后ＭＧＭＴｍＲＮＡ水平、ＭＧＭＴ活性及其对ＡＣＮＵ抗药性的变化．发现针对ＭＧＭＴｍＲＮＡ５’端的反义ＲＮＡ能够有效地降低ＭＧＭＴｍＲＮＡ水平和ＭＧＭＴ活性并使细胞对ＡＣＮＵ的敏感性提高４．６倍;针对ＭＧＭＴｍＲＮＡ全长的反义ＲＮＡ也能在一定程度上调节细胞的ＭＧＭＴｍＲＮＡ水平和ＭＧＭＴ活性并增加细胞对ＡＣＮＵ的敏感性,而针对３’端序列的反义ＲＮＡ对ＭＧＭＴ活性没有调节作用．     

Antisense therapy of hepatitis B virus infection

Chronic infection with the hepatitis B virus (HBV) is a major health problem worldwide. The only established therapy is interferon-a with an efficacy of only 30–40% in highly selected patients. The discovery of animal viruses closely related to the HBV has contributed to active research on antiviral therapy of chronic hepatitis B. The animal model tested and described in this article are Peking ducks infected with the duck hepatitis B virus (DHBV). Molecular therapeutic strategies aimed at blocking gene expression include antisense DNA. An antisense oligodeoxynucleotide directed against the 5′-region of the preS gene of DHBV inhibited viral replication and gene expression in vitro in primary duck hepatocytes and in vivo in Peking ducks. These results demonstrate the potential clinical use of antisense DNA as antiviral therapeutics.     

Determination of the Solution Conformation of HIV-1 Tat(1–9) Peptides by Means of Molecular Dynamics Simulations Considering NMR Data and Docking Studies into an Active Site Model of DP IV

The human immunodeficiency virus 1 Tat protein suppresses antigen-, anti-CD3-and mitogen-induced activation of human T cells when added to T cell cultures. This activity is important for the development of AIDS because lymphocytes from HIV-infected individuals exhibit a similar antigen-specific dysfunction. Moreover, Tat was found to interact with dipeptidyl peptidase IV (DP IV). To find out the amino acid sequence important for the inhibition of the DP IV enzymatic activity we investigated N-terminal Tat(1–9) peptide analogues with amino acid substitutions in different positions. Interestingly, the exchange of Pro⁶ with Leu and Asp⁵ with Ile strongly diminished the DP IV inhibition by Tat(1–9). Based on data derived from one-and two-dimensional ¹H NMR investigations the solution conformations of the three nonapeptides in water were determined by means of molecular dynamics simulations. These conformations were used for studies of the docking behavior of the peptides into a model of the active site of DP IV. The results suggest that several attractive interactions between the native Tat(1–9) and DP IV lead to a stable complex and that the reduced affinity of both L⁶-Tat(1–9) and I⁵-Tat(1–9) derivatives might be caused by conformational alterations in comparison to the parent peptide.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089480040200     

Bacteriophage and bacteriophage resistance in lactic acid bacteria

Abstract: The study of bacteriophage-host interactions has been instrumental in the development of genetic systems in many genera, and laid many of the foundations of modern molecular genetics. Research into bacteriophage and bacteriophage resistance in the lactic acid bacteria has moved into a new and exciting dimension in recent years. Mechanisms such as adsorption inhibition, restriction and modification, and abortive infection which have been detected and described phenotypically over the past decade are now being subjected to molecular analysis, and this has led to a better understanding of the nature and variety of resistance systems employed by lactic acid bacteria to combat phage attack. In addition, analysis of different bacteriophage has increased our knowledge of these ubiquitous particles to the point where it is possible to construct novel phage resistances based on the phage genome itself. This review outlines the recent progress in the molecular analysis of bacteriophage, bacteriophage resistance and counter resistance, and the construction of novel resistance mechanisms.