5-脱氧杂氮胞苷抑制小鼠附植前的胚胎发育

DNA甲基化在哺乳动物发育过程中有关键作用．在小鼠附植前胚胎发育过程中,DNA甲基化一直处于动态变化过程中．通过将体外受精胚在5-AZA-CdR中持续培养,研究5-AZA-CdR对小鼠附植前胚胎发育的影响,为附植前胚胎发育机理的研究及5-AZA-CdR的毒副作用研究提供试验基础．从原核期加入不同浓度的5-AZA-CdR时,胚胎不能发育到桑椹胚(0.2 和1.0 μmol/L)和4-细胞胚(5.0 μmol/L);从2-细胞期加入时,胚胎阻滞于未致密化的8-细胞(0.2 和1.0 μmol/L)和3/4-细胞期(5.0 μmol/L);而当从4-细胞加入时,虽然胚胎能够发育到早期桑椹胚,但发育比例同对照相比显著降低(P < 0.05)．进一步检测凋亡、基因组DNA甲基化和整体转录活性,结果显示,高浓度的5-AZA-CdR导致8-细胞和早期桑椹胚发生早期凋亡,而低浓度的5-AZA-CdR引起8-细胞和早期桑椹胚基因组DNA甲基化的降低和转录活性的降低,并且这种降低呈浓度依赖性．所以加入低浓度的5-AZA-CdR时,胚胎的DNA甲基化降低,引起转录活性的降低,进而导致胚胎发育的停滞．     

An affinity-enhanced neutralizing antibody against the membrane-proximal external region of human immunodeficiency virus type 1 gp41 recognizes an epitope between those of 2F5 and 4E10

The membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 bears the epitopes of two broadly neutralizing antibodies (Abs), 2F5 and 4E10, making it a target for vaccine design. A third Ab, Fab Z13, had previously been mapped to an epitope that overlaps those of 2F5 and 4E10 but only weakly neutralizes a limited set of primary isolates. Here, libraries of Fab Z13 variants displayed on phage were engineered and affinity selected against an MPER peptide and recombinant gp41. A high-affinity variant, designated Z13e1, was isolated and found to be approximately 100-fold improved over the parental Fab not only in binding affinity for the MPER antigens but also in neutralization potency against sensitive HIV-1. Alanine scanning of MPER residues 664 to 680 revealed that N671 and D674 are crucial for peptide recognition as well as for the neutralization of HIV-1 by Z13e1. Ab competition studies and truncation of MPER peptides indicate that Z13e1 binds with high affinity to an epitope between and overlapping with those of 2F5 and 4E10, with the minimal peptide epitope WASLWNWFDITN. Still, Z13e1 remained about an order of magnitude less potent than 4E10 against several isolates of pseudotyped HIV-1. The sum of our molecular analyses with Z13e1 suggests that the segment on the MPER of gp41 between the 2F5 and 4E10 epitopes is exposed on the functional envelope trimer but that access to the specific Z13e1 epitope within this segment is limited. Thus, the ability of MPER-bearing immunogens to elicit potent HIV-1-neutralizing Abs may depend in part on recapitulating the particular constraints that the functional envelope trimer imposes on the segment of the MPER to which Z13e1 binds.     

Identifying Hendra virus diversity in pteropid bats

Hendra virus (HeV) causes a zoonotic disease with high mortality that is transmitted to humans from bats of the genus Pteropus (flying foxes) via an intermediary equine host. Factors promoting spillover from bats to horses are uncertain at this time, but plausibly encompass host and/or agent and/or environmental factors. There is a lack of HeV sequence information derived from the natural bat host, as previously sequences have only been obtained from horses or humans following spillover events. In order to obtain an insight into possible variants of HeV circulating in flying foxes, collection of urine was undertaken in multiple flying fox roosts in Queensland, Australia. HeV was found to be geographically widespread in flying foxes with a number of HeV variants circulating at the one time at multiple locations, while at times the same variant was found circulating at disparate locations. Sequence diversity within variants allowed differentiation on the basis of nucleotide changes, and hypervariable regions in the genome were identified that could be used to differentiate circulating variants. Further, during the study, HeV was isolated from the urine of flying foxes on four occasions from three different locations. The data indicates that spillover events do not correlate with particular HeV isolates, suggesting that host and/or environmental factors are the primary determinants of bat-horse spillover. Thus future spillover events are likely to occur, and there is an on-going need for effective risk management strategies for both human and animal health.     

Genome sequencing and comparison of two nonhuman primate animal models, the cynomolgus and Chinese rhesus macaques

The nonhuman primates most commonly used in medical research are from the genus Macaca. To better understand the genetic differences between these animal models, we present high-quality draft genome sequences from two macaque species, the cynomolgus/crab-eating macaque and the Chinese rhesus macaque. Comparison with the previously sequenced Indian rhesus macaque reveals that all three macaques maintain abundant genetic heterogeneity, including millions of single-nucleotide substitutions and many insertions, deletions and gross chromosomal rearrangements. By assessing genetic regions with reduced variability, we identify genes in each macaque species that may have experienced positive selection. Genetic divergence patterns suggest that the cynomolgus macaque genome has been shaped by introgression after hybridization with the Chinese rhesus macaque. Macaque genes display a high degree of sequence similarity with human disease gene orthologs and drug targets. However, we identify several putatively dysfunctional genetic differences between the three macaque species, which may explain functional differences between them previously observed in clinical studies.     

Effect of a traditional Chinese medicine preparation Xindi soft capsule on rat model of acute blood stasis: a urinary metabonomics study based on liquid chromatography-mass spectrometry

Xindi soft capsule is a traditional Chinese medicine preparation which consists of sea buckthorn flavonoids and sea buckthorn berry oil. In this study, a urinary metabonomics method based on the ultra-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS) was used to evaluate the efficacy and study the mechanism of traditional Chinese medicine preparation to blood stasis. With pattern recognition analysis (principal component analysis and partial least squares-discriminate analysis) of urinary metabolites, a clear separation of acute blood stasis model group and healthy control group was achieved, the dose groups were located between acute blood stasis model group and healthy control group showing a tendency of recovering to healthy control group, high dose and middle dose were more effective than low dose. Some significantly changed metabolites like cholic acid, phenylalanine and kynurenic acid have been found and identified and used to explain the mechanism. The work shows that the metabonomics method is a valuable tool in the research mechanism of traditional Chinese medicine.     

Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitro

Members of the RNase III family are the primary cellular agents of dsRNA (double-stranded RNA) processing. Bacterial RNases III function as homodimers and contain two dsRBDs (dsRNA-binding domains) and two catalytic sites. The potential for functional cross-talk between the catalytic sites and the requirement for both dsRBDs for processing activity are not known. It is shown that an Escherichia coli RNase III heterodimer that contains a single functional wt (wild-type) catalytic site and an inactive catalytic site (RNase III[E117A/wt]) cleaves a substrate with a single scissile bond with a k(cat) value that is one-half that of wt RNase III, but exhibits an unaltered K(m). Moreover, RNase III[E117A/wt] cleavage of a substrate containing two scissile bonds generates singly cleaved intermediates that are only slowly cleaved at the remaining phosphodiester linkage, and in a manner that is sensitive to excess unlabelled substrate. These results demonstrate the equal probability, during a single binding event, of placement of a scissile bond in a functional or nonfunctional catalytic site of the heterodimer and reveal a requirement for substrate dissociation and rebinding for cleavage of both phosphodiester linkages by the mutant heterodimer. The rate of phosphodiester hydrolysis by RNase III[E117A/wt] has the same dependence on Mg(2+) ion concentration as that of the wt enzyme, and exhibits a Hill coefficient (h) of 2.0+/-0.1, indicating that the metal ion dependence essentially reflects a single catalytic site that employs a two-Mg(2+)-ion mechanism. Whereas an E. coli RNase III mutant that lacks both dsRBDs is inactive, a heterodimer that contains a single dsRBD exhibits significant catalytic activity. These findings support a reaction pathway involving the largely independent action of the dsRBDs and the catalytic sites in substrate recognition and cleavage respectively.     

Molecular evolution of glutathione peroxidase (<Emphasis Type="Italic">Gpx</Emphasis>) family genes in desert poplar (<Emphasis Type="Italic">Populus euphratica</Emphasis> Oliv.)

Populus euphratica Oliv. is a poplar species that is distributed mainly in deserts, making it an interesting model in which to investigate molecular mechanisms underlying different stress responses. Here, we used molecular population genetic methods to detect potential selection in candidate genes belonging to the P. euphratica glutathione (GSH) peroxidase (Gpx) family, which are associated with an enzymatic mechanism that combats oxidative damage caused by reactive oxygen species (ROS) in plant cells; earlier studies have shown that Gpx proteins play important roles in coping with increased ROS levels during biotic and abiotic stresses in plants. We analyzed the nucleotide diversity and divergence patterns of five loci encoding Gpx genes, and 16 reference loci used as controls, in order to detect departures from the neutral expectation. Gpx1 has an excess of mid-frequency alleles; high intraspecific nucleotide diversity, distributed in the upper tail of the simulated neutral model; and extensive LD, showing strong evidence of balancing selection/local adaptation. The Gpx3.2 gene exhibits very low nucleotide diversity and divergence, suggesting that it has evolved under strong purifying selection. We failed to detect any evidence for natural selection at the other loci (Gpx2, Gpx4, and Gpx5) compared with the reference loci. The results show that nucleotide diversity and/or divergence differ greatly between members of the Gpx gene family, resulting from differential selective pressure acting on genes, and that adaptive evolution influenced the distribution of P. euphratica in desert regions.