Improved gene expression in resting macrophages using an oligopeptide derived from Vpr of human immunodeficiency virus type-1

Vpr, an accessory gene product of human immunodeficiency virus type-1, is thought to transport a viral DNA from the cytoplasm to the nucleus in resting macrophages. Previously, we reported that a peptide encompassing amino acids 52-78 of Vpr (C45D18) promotes the nuclear trafficking of recombinant proteins that are conjugated with C45D18. Here, we present evidence that C45D18, when conjugated with a six-branched cationic polymer of poly(N,N-dimethylaminopropylacrylamide)-block-oligo(4-aminostyrene) (SV: star vector), facilitates gene expression in resting macrophages. Although there was no difference between SV alone and C45D18-SV with respect to gene transduction into growing cells, C45D18-SV resulted in more than 40-fold greater expression of the exogenous gene upon transduction into chemically differentiated macrophages and human quiescent monocyte-derived macrophages. The data suggest that C45D18 contributes to improving the ability of a non-viral vector to transduce macrophages with exogenous genes and we discuss its further application.     

Regulation of cellular gene expression and function by the human immunodeficiency virus type 1 tat protein

The human immunodeficiency virus type 1 Tat protein is a potent activator of viral gene expression and replication. Tat can also affect the expression of cellular genes including cytokines, extracellular matrix proteins, enzymes degrading the basement membrane and cell cycle-related proteins, and can regulate cellular functions such as growth, migration and angiogenesis. In addition, under certain circumstances, Tat may have tumorigenic effects. These activities of Tat appear to be mediated by different mechanisms such as the transactivation of cellular gene expression or the interaction of extracellular Tat with the cell membrane through both receptor-mediated and nonreceptor-mediated interactions. Deregulation of cellular gene expression and function by Tat cause abnormalities which may participate in AIDS pathogenesis and in the development of AIDS-associated disorders.     

Novel aspects of the regulation of a cDNA (Arf1) from Chlamydomonas with high sequence identity to animal ADP-ribosylation factor 1

ADP-ribosylation factor (ARF) is a highly conserved, low molecular mass (ca. 21 kDa) GTP-binding protein that has been implicated in vesicle trafficking and signal transduction in yeast and mammalian cells. However, little is known of ARF in plant systems. A putative ARF polypeptide was identifed in subcellular fractions of the green alga Chlamydomonas reinhardtii, based on [³²P]GTP binding and immunoblot assays. A cDNA clone was isolated from Chlamydomonas (Arf1), which encodes a 20.7 kDa protein with 90% identity to human ARF1. Northern blot analyses showed that levels of Arf1 mRNA are highly regulated during 12 h/12 h light/dark (LD) cycles. A biphasic pattern of expression was observed: a transient peak of Arf1 mRNA occurred at the onset of the light period, which was followed ca. 12 h later by a more prominent peak in the early to mid-dark period. When LD-synchronized cells were shifted to continuous darkness, the dark-specific peak of Arf1 mRNA persisted, indicative of a circadian rhythm. The increase in Arf1 mRNA at the beginning of the light period, however, was shown to be light-dependent, and, moreover, dependent on photosynthesis, since it was prevented by DCMU. We conclude that the biphasic pattern of Arf1 mRNA accumulation during LD cycles is due to regulation by two different factors, light (which requires photosynthesis) and the circadian clock. Thus, these studies identify a novel pattern of expression for a GTP-binding protein gene.     

Suppressive effect of elongation factor 2 on apoptosis induced by HIV-1 viral protein R

Rapid CD4+ lymphocyte depletion due to cell death caused by HIV infection is one of the hallmarks of acquired immunodeficiency syndrome. HIV-1 viral protein R (Vpr) induces apoptosis and is believed to contribute to CD4+ lymphocyte depletion. Thus, identification of cellular factors that potentially counteract this detrimental viral effect will not only help us to understand the molecular action of Vpr but also to design future antiviral therapies. In this report, we describe identification of elongation factor 2 (EF2) as such a cellular factor. Specifically, EF2 protein level is responsive to vpr gene expression; it is able to suppress Vpr-induced apoptosis when it is overproduced beyond its physiological level. EF2 was initially identified through a genome-wide multicopy suppressor search for Vpr-induced apoptosis in a fission yeast model system. Overproduction of fission yeast Ef2 completely abolishes Vpr-induced cell killing in fission yeast. Similarly, overexpression of the human homologue of yeast Ef2 in a neuroblastoma SKN-SH cell line and two CD4+ H9 and CEM-SS T-cell lines also blocked Vpr-induced apoptosis. The anti-apoptotic property of EF2 is demonstrated by its ability to suppress caspase 9 and caspase 3-mediated apoptosis induced by Vpr. In addition, it also reduces cytochrome c release induced by Vpr, staurosporine and TNFα. The fact that overproduction of EF2 blocks Vpr-induced cell death both in fission yeast and human cells, suggested that EF2 posses a highly conserved anti-apoptotic activity. Moreover, the responsive elevation of EF2 to Vpr suggests a possible host innate antiviral response.     

中华蜜蜂信息素结合蛋白ASP1 cDNA的克隆及时空表达

信息素结合蛋白（pheromone binding proteins, PBPs）在昆虫信息素的识别、传递和处理过程中具有重要作用。本研究首次克隆了中华蜜蜂Apis cerana cerana的一个PBP基因Ac-ASP1（GenBank序列号为DQ449670）,其预测蛋白具有典型的气味结合蛋白（OBPs）标志（即成熟肽含有6个保守的半胱氨酸）。利用real-time PCR技术对Ac-ASP1在中蜂不同组织和发育历期的时空表达谱进行了鉴定。绝对定量结果显示Ac-ASP1高丰度地表达于工蜂触角（2.07×10⁶ 拷贝数/μg）,而在其他组织（如头、胸、腹、翅及足）中呈低丰度表达（10²拷贝数/μg）; 相对定量结果显示Ac-ASP1在各发育历期如幼虫、蛹以及成虫发育早期（1～6日龄）均有大量表达,而在21日龄前后具有另外一个高丰度表达时期。这些结果可为明确Ac-ASP1在中蜂蜂王信息素信号识别传递过程中的作用提供参考。     

Site-specific integration of retroviral DNA in human cells using fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc-finger protein E2C

During the life cycle of retroviruses, establishment of a productive infection requires stable joining of a DNA copy of the viral RNA genome into host cell chromosomes. Retroviruses are thus promising vectors for the efficient and stable delivery of genes in therapeutic protocols. Integration of retroviral DNA is catalyzed by the viral enzyme integrase (IN), and one salient feature of retroviral DNA integration is its lack of specificity, as many chromosomal sites can serve as targets for integration. Despite the promise for success in the clinic, one major drawback of the retrovirus-based vector is that any unintended insertion events from the therapy can potentially lead to deleterious effects in patients, as demonstrated by the development of malignancies in both animal and human studies. One approach to directing integration into predetermined DNA sites is fusing IN to a sequence-specific DNA-binding protein, which results in a bias of integration near the recognition site of the fusion partner. Encouraging results have been generated in vitro and in vivo using fusion protein constructs of human immunodeficiency virus type 1 IN and E2C, a designed polydactyl zinc-finger protein that specifically recognizes an 18-base pair DNA sequence. This review focuses on the method for preparing infectious virions containing the IN fusion proteins and on the quantitative PCR assays for determining integration site specificity. Efforts to engineer IN to recognize specific target DNA sequences within the genome may lead to development of effective retroviral vectors that can safely deliver gene-based therapeutics in a clinical setting.     

Differential regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) by phosphorylation directed by the cyclin encoded by Murine Herpesvirus 68

Members of the gamma2-herpesvirus family encode cyclin-like proteins that have the ability to deregulate mammalian cell cycle control. Here we report the key features of the viral cyclin encoded by Murine Herpesvirus 68, M cyclin. M cyclin preferentially associated with and activated cdk2; the M cyclin/cdk2 holoenzyme displayed a strong reliance on phosphorylation of the cdk T loop for activity. cdk2 associated with M cyclin exhibited substantial resistance to the cdk inhibitor proteins p21(Cip) and p27(Kip). Furthermore, M cyclin directed cdk2 to phosphorylate p27(Kip1) on threonine 187 (T187) and cellular expression of M cyclin led to down-regulation of p27(Kip1) and the partial subversion of the associated G1 arrest. Mutation of T187 to a non-phosphorylatable alanine rendered the p27(Kip1)-imposed G1 arrest resistant to M cyclin expression. Unlike the related K cyclin, M cyclin was unable to circumvent the G1 arrest associated with p21(Cip1) and was unable to direct its associated catalytic subunit to phosphorylate this cdk inhibitor. These results imply that M cyclin has properties that are distinct from other viral cyclins and that M cyclin expression alone is insufficient for S phase entry.     

东亚飞蝗fem-1基因的克隆与表达分析

秀丽隐杆线虫Caenorhabditis elegans fem-1基因是性别决定的关键基因。本研究基于生物信息学方法从东亚飞蝗Locusta migratoria manilensis的转录组数据库中克隆出了线虫fem-1的3个同源基因, 将其分别命名为Lmfem-1a, Lmfem-1b和Lmfem-1c (GenBank登录号分别为AB698670, AB698671和AB698672)。其cDNA序列长度分别为2 233, 2 625和2 142 bp, 分别编码662, 642和638个氨基酸。生物信息学分析显示, Lmfem-1a, Lmfem-1b和Lmfem-1c分别含有6, 8和8个典型的锚蛋白重复序列模体。组织表达谱分析发现, Lmfem-1a, Lmfem-1b和Lmfem-1c基因在检测的所有组织中都有表达, 但均在精巢中的表达水平最高, 说明Lmfem-1a, Lmfem-1b和Lmfem-1c基因可能参与东亚飞蝗的多种生理过程, 并受到严格的表达调控。而且, 随着精巢的发育, Lmfem-1a, Lmfem-1b和Lmfem-1c的表达均逐渐增强, 可能与东亚飞蝗的精子形成有关, 但这3个基因是否参与东亚飞蝗的性别决定还有待进一步研究。     

Leishmania (Viannia) panamensis: cloning of the histone H1 genes by representational difference analysis

We report the use of representational difference analysis to identify genes that have up-regulated expression in the amastigote life-cycle stage of Leishmania (Viannia) panamensis. This simultaneous process of selection and amplification allowed the cloning of several specific DNA fragments. One of them shows a high percentage of similarity with histone H1 genes from other Trypanosomatids and, as expected, is up-regulated in the amastigote life-cycle stage. The gene is present in two copies that are expressed at different levels in promastigotes and also in amastigotes, which seems to be a consequence of their different 3' untranslated regions.     

烟粉虱MEAM1隐种漆酶-1基因全长cDNA克隆、序列分析与组织表达

多酚氧化酶是多种刺吸式昆虫唾液中的关键酶类,可以通过干扰寄主植物正常的氧化还原反应来调控食物来源使其更有利于昆虫本身。昆虫体内的漆酶-1属于多酚氧化酶家族,被认为参与昆虫取食过程中的金属离子代谢、降解植物次生有毒物质以及免疫防御等生理活动。为研究漆酶-1基因在烟粉虱MEAM1隐种Bemisia tabaci Middle East-Asia Minor 1组织中的功能,利用RACE技术首次获得了烟粉虱MEAMI隐种漆酶-1（laccase-1,lac-1）基因的cDNA全长序列,命名为Btlac-1 ( GenBank 登录号： JQ966215）。结果表明,该基因含有一个2 733 bp的开放阅读框,编码910个氨基酸,其编码产物含有3个Cu-oxidase功能域, 属于蓝多铜氧化酶家族成员。同源性比较分析表明,Btlac-1与黑尾叶蝉Nephotettix cincticeps和豌豆蚜Acyrthosiphon pisum的lac-1编码的氨基酸序列一致性达58%。Btlac-1在不同组织和发育时期的转录表达分析表明, Btlac-1在烟粉虱MEAM1隐种中肠中的转录水平最高,显著高于头胸部和腹部组织中,且在各个发育时期均有表达,在成虫期的表达水平较高。该结果为进一步明确lac-1在烟粉虱取食过程的生理作用奠定基础,也为探讨刺吸式昆虫与植物相互作用机制提供理论依据。     

Gene expression analysis in LLC-PK1 renal tubular cells by atrial natriuretic peptide (ANP): correlation of homologous human genes with renal response

We used human DNA microarray to explore the differential gene expression profiling of atrial natriuretic peptide (ANP)-stimulated renal tubular epithelial kidney cells (LLC-PK1) in order to understand the biological effect of ANP on renal kidney cell's response. Gene expression profiling revealed 807 differentially expressed genes, consisting of 483 up-regulated and 324 down-regulated genes. The bioinformatics tool was used to gain a better understanding of differentially expressed genes in porcine genome homologous with human genome and to search the gene ontology and category classification, such as cellular component, molecular function and biological process. Four up-regulated genes of ATP1B1, H3F3A, ITGB1 and RHO that were typically validated by real-time quantitative PCR (RT-qPCR) analysis serve important roles in the alleviation of renal hypertrophy as well as other related effects. Therefore, the human array can be used for gene expression analysis in pig kidney cells and we believe that our findings of differentially expressed genes served as genetic markers and biological functions can lead to a better understanding of ANP action on the renal protective system and may be used for further therapeutic application.     

Babesia bovis: the development of an expression oligonucleotide microarray

The availability of a stage-specific Babesia bovis expression profile can facilitate the identification of candidate vaccine antigens. In addition, highly expressed genes during a particular developmental stage may suggest their relevance during that stage. In this study, we generated and validated a custom B. bovis high density oligonucleotide microarray that can be used to examine gene expression levels. An expression profile of in vitro cultured intraerythrocytic stage genes that could be distinguished from contaminating host message was established, and the expression levels of over 1000 genes were ranked. Ranking order was validated using quantitative real time PCR on a twelve randomly selected open reading frames whose expression levels range from the highest to the acceptable lowest. Expression of annotated ORFs was consistent with results from a recently published B. bovis expression sequence tag study. Therefore, we conclude that the microarray is suitable for analyzing B. bovis gene expression, and present the complete B. bovis infected erythrocyte expression profile.