Evaluation of replication and pathogenicity of avian influenza a H7 subtype viruses in a mouse model

Avian influenza A H7 subtype viruses pose a significant threat to human health because of their ability to transmit directly from domestic poultry to humans and to cause disease and, sometimes, death. Although it is important to develop vaccines against viruses of this subtype, very limited information is available on the immune response and pathogenesis of H7 viruses in animal models such as mice and ferrets. Ten H7 viruses were selected for possible vaccine development on the basis of their phylogenetic relationships and geographical locations. The virulence of the 10 viruses for mice and the immunogenicity of the viruses in mice and ferrets were evaluated to study the extent of antigenic relatedness and the level of cross-reactivity of antibodies. Most of the viruses showed similar patterns of cross-reactivity with mouse and ferret antisera. The Eurasian viruses elicited broadly cross-reactive antibodies that neutralized viruses from both Eurasian and North American lineages, but the converse was not true. A subset of the viruses was also evaluated for the ability to replicate and cause disease in BALB/c mice following intranasal administration. H7 subtype viruses were able to infect mice without adaptation and manifested different levels of lethality and kinetics of replication. On the basis of phylogenetic data, induction of broadly cross-neutralizing antibodies in mouse and ferret antisera, and their ability to replicate in mice, we have selected A/Netherlands/219/03 (subtype H7N7) and A/chicken/BC/CN-7/04 (subtype H7N3) viruses for vaccine development. The mouse model can be used for the preclinical evaluation of these vaccines against H7 subtype viruses.     

Rapid reversion of sequence polymorphisms dominates early human immunodeficiency virus type 1 evolution

The error-prone replication of human immunodeficiency virus type 1 (HIV-1) enables it to continuously evade host CD8+ T-cell responses. The observed transmission, and potential accumulation, of CD8+ T-cell escape mutations in the population may suggest a gradual adaptation of HIV-1 to immune pressures. Recent reports, however, have highlighted the propensity of some escape mutations to revert upon transmission to a new host in order to restore efficient replication capacity. To more specifically address the role of reversions in early HIV-1 evolution, we examined sequence polymorphisms arising across the HIV-1 genome in seven subjects followed longitudinally 1 year from primary infection. As expected, numerous nonsynonymous mutations were associated with described CD8+ T-cell epitopes, supporting a prominent role for cellular immune responses in driving early HIV-1 evolution. Strikingly, however, a substantial proportion of substitutions (42%) reverted toward the clade B consensus sequence, with nearly one-quarter of them located within defined CD8 epitopes not restricted by the contemporary host's HLA. More importantly, these reversions arose significantly faster than forward mutations, with the most rapidly reverting mutations preferentially arising within structurally conserved residues. These data suggest that many transmitted mutations likely incur a fitness cost that is recovered through retrieval of an optimal, or ancestral, form of the virus. The propensity of mutations to revert may limit the accumulation of immune pressure-driven mutations in the population, thus preserving critical CD8+ T-cell epitopes as vaccine targets, and argue against an unremitting adaptation of HIV-1 to host immune pressures.     

Micro RNA 145 targets the insulin receptor substrate-1 and inhibits the growth of colon cancer cells

The insulin receptor substrate-1 (IRS-1), a docking protein for both the type 1 insulin-like growth factor receptor (IGF-IR) and the insulin receptor, is known to send a mitogenic, anti-apoptotic, and anti-differentiation signal. Several micro RNAs (miRs) are suggested by the data base as possible candidates for targeting IRS-1. We show here that one of the miRs predicted by the data base, miR145, whether transfected as a synthetic oligonucleotide or expressed from a plasmid, causes down-regulation of IRS-1 in human colon cancer cells. IRS-1 mRNA is not decreased by miR145, while it is down-regulated by an siRNA targeting IRS-1. Targeting of the IRS-1 3'-untranslated region (UTR) by miR145 was confirmed using a reporter gene (luciferase) expressing the miR145 binding sites of the IRS-1 3'-UTR. In agreement with the role of IRS-1 in cell proliferation, we show that treatment of human colon cancer cells with miR145 causes growth arrest comparable to the use of an siRNA against IRS-1. Taken together, these results identify miR145 as a micro RNA that down-regulates the IRS-1 protein, and inhibits the growth of human cancer cells.     

大肠杆菌Ee株SLT-IIeB与FedF基因的融合表达及表达产物的免疫原性研究

根据猪水肿病大肠杆菌Ee株主要免疫原性片段SLT-IIeB和FedF的基因序列,利用PCR技术克隆目的片段,将SLT-IIeB和FedF基因依序串联于谷胱甘肽-S-转移酶(GST)表达系统的GST下游,在大肠杆菌中成功获得了大小约为63kDa融合蛋白GST-SF,Western blot检测证实表达的融合蛋白具有良好的生物学活性。以纯化的融合蛋白为抗原免疫新西兰兔制备血清,体外活性试验表明,所制备的抗血清能中和Ee株水肿毒素对Vero细胞的病变效应;黏附抑制试验证实抗血清能抑制Ee株对猪小肠刷状缘细胞的黏附。将融合蛋白免疫小鼠,结果表明,GST-SF能够诱发较好的免疫反应,并能提供对Ee株5LD50致死剂量的70%保护率。研究表明GST-SF融合蛋白具有良好的免疫原性,具有良好的应用前景。     

腺嘌呤对大肠杆菌DH5α及其耐乙酸突变株DA19生长和代谢的影响

大肠杆菌DH5α和DA19在碳源限制的基本培养基中进行分批培养和氮源限制的基本培养基中进行连续培养,发现添加腺嘌呤会使DH5α生长改善,乙酸产生减少;对DA19的生长和代谢影响不明显。连续培养细胞的蛋白质组分析结果显示:添加腺嘌呤后DA19 purH表达水平降低,而DH5α purH表达水平不变,表明DH5α嘌呤核苷酸从头合成能力差;DH5α yddS表达上调,gnd表达水平稍有提高,细胞生长改善。     

非洲狼尾草未受精无孢子生殖胚囊退化研究

为理解植物无孢子生殖胚囊未受精条件下的退化,对无孢子生殖植物非洲狼尾草未受精成熟胚囊中央细胞退化做了细胞形态学研究。没有受精的中央细胞退化时最显著的特点是细胞核产生核膜囊泡。核膜囊泡有两种类型:单层膜的囊泡和双层膜的囊泡,单层膜囊泡在细胞质中,双层膜囊泡在细胞核内。核膜囊泡有两种发生方式:1)核膜的外膜向细胞质一侧膨胀产生囊泡,囊泡进入细胞质;2)核膜向核内凹陷形成囊泡,囊泡进入细胞核。核膜囊泡类型与产生方式密切关联。核膜囊泡吞噬并消化包括线粒体在内的细胞质和核质。     

Cdc42和球形肌动蛋白在卵母细胞胞质分裂中的定位分析

研究活性Cdc42与球形肌动蛋白(G-actin)在爪蟾卵母细胞胞质分裂中的定位关系。分别用GFP-wGBDmRNA与罗丹明-594-微管蛋白、Alexa-488-球形肌动蛋白与罗丹明-594-微管蛋白、GFP-wGBDmRNA与Alexa-594-球形肌动蛋白共同显微注射爪蟾卵母细胞。利用共聚焦显微镜,时间延迟摄影方法,分别观察活体卵母细胞中活性Cdc42、球形肌动蛋白在胞质分裂过程中的定位,以及活性Cdc42与球形肌动蛋白在胞质分裂中的定位关系。在卵母细胞胞质分裂中,活性Cdc42与球形肌动蛋白存在空间上共定位现象,并且在时相上具有一致性。结果提示活性Cdc42和球形肌动蛋白在卵母细胞胞质分裂过程中密切相关。