PCR-based generation of shRNA libraries from cDNAs

Background  

The use of small interfering RNAs (siRNAs) to silence target gene expression has greatly facilitated mammalian genetic analysis by generating loss-of-function mutants. In recent years, high-throughput, genome-wide screening of siRNA libraries has emerged as a viable approach. Two different methods have been used to generate short hairpin RNA (shRNA) libraries; one is to use chemically synthesized oligonucleotides, and the other is to convert complementary DNAs (cDNAs) into shRNA cassettes enzymatically. The high cost of chemical synthesis and the low efficiency of the enzymatic approach have hampered the widespread use of screening with shRNA libraries.     

LSH catalyzes ATP-driven exchange of histone variants macroH2A1 and macroH2A2

LSH, a homologue of the ISWI/SNF2 family of chromatin remodelers, is required in vivo for deposition of the histone variants macroH2A1 and macroH2A2 at specific genomic locations. However, it remains unknown whether LSH is directly involved in this process or promotes other factors. Here we show that recombinant LSH interacts in vitro with macroH2A1–H2B and macroH2A2–H2B dimers, but not with H2A.Z–H2B dimers. Moreover, LSH catalyzes the transfer of macroH2A into mono-nucleosomes reconstituted with canonical core histones in an ATP dependent manner. LSH requires the ATP binding site and the replacement process is unidirectional leading to heterotypic and homotypic nucleosomes. Both variants macroH2A1 and macroH2A2 are equally well incorporated into the nucleosome. The histone exchange reaction is specific for histone variant macroH2A, since LSH is not capable to incorporate H2A.Z. These findings define a previously unknown role for LSH in chromatin remodeling and identify a novel molecular mechanism for deposition of the histone variant macroH2A.     

Research on the feeding behavior of Neospastis simaona larvae

本文对思茅新木蛾Neospastis simaona Wang幼虫的取食行为、取食偏好性以及明暗条件对其取食量的影响进行了观察研究,为该昆虫的防治提供了理论依据.研究结果表明:思茅新木蛾幼虫随着龄期的增加,取食次数增多,取食总时间增加,但一次取食时间无明显变化;思茅新木蛾幼虫对木荷(Schima superba)有强烈嗜食性,4龄幼虫喜食木荷幼叶,5、6龄幼虫对木荷幼叶、成熟叶无明显偏好性;明暗条件对思茅新木蛾4至6龄幼虫的取食量没有显著影响(P＞0.05).     

Phylogenetics of early branching eudicots: Comparing phylogenetic signal across plastid introns, spacers, and genes

Recent phylogenetic analyses revealed a grade with Ranunculales,Sabiales,Proteales,Trochodendrales,and Buxales as first branching eudicots,with the respective positions of Proteales and Sabiales still ...     

β(1) -adrenergic receptor autoantibodies from heart failure patients enhanced TNF-α secretion in RAW264.7 macrophages in a largely PKA-dependent fashion

Autoantibodies against the second extracellular loop of β₁‐adrenergic receptor (β₁‐AA) not only contribute to increased susceptibility to heart failure, but also play a causative role in myocardial remodeling through their catecholamine‐like effects via binding with the β₁‐adrenergic receptor. The current study was designed to determine whether β₁‐AA isolated from the sera of heart failure patients could cause TNF‐α secretion from the murine macrophage‐like cell line RAW264.7. Blood samples were collected from 40 patients who had suffered heart failure, as well as from 40 healthy subjects. The titer of β₁‐AA and the level of TNF‐α were detected using ELISA. The effect of β₁‐AA on murine macrophage‐like cell line RAW264.7 proliferation was detected by CCK‐8 kits and CFSE assay. Western blot assay was used to analyze the expression of phospho‐VASP. β₁‐AA appeared more frequently in patients with heart failure than in healthy subjects. The β₁‐AA isolated from heart failure patients promoted an increase of TNF‐α levels, which could be completely blocked by the selective β₁‐adrenergic receptor antagonist metoprolol and the second extracellular loop of β₁‐adrenergic receptor (β₁‐AR‐EC_II), but only partially inhibited by PKA inhibitor H89. Furthermore, the β₁‐AA could enhance the proliferation of RAW264.7 cells in vitro. Meanwhile, the expression of phospho‐VASP was markedly increased in the presence of β₁‐AA. These results demonstrate for the first time that the β₁‐AA isolated from heart failure patients could bind with β₁‐AR on the surface of RAW264.7 cells, causing the release of TNF‐α largely in a PKA‐dependent fashion. J. Cell. Biochem. 113: 3218–3228, 2012. © 2012 Wiley Periodicals, Inc.     

Cloning and expression pattern of heat shock protein genes from the endoparasitoid wasp, Pteromalus puparum in response to environmental stresses

Six heat shock protein (HSP) genes from five HSP families in the parasitoid, Pteromalus puparum, were evaluated for their response to temperature (-15 ~ 3°C , and 30 ~ 42°C for 1 h), heavy metals (0.5 ~ 5 mM Cd(2+) and Cu(2+) for 24 h and 60 h), and starvation (24 h). Compared with other insect HSPs, all conserved motifs are found in P. puparum HSPs, and they are very similar to those of the recently sequenced ectoparasitoid Nasonia vitripennis. The temporal gene expression patterns indicated that these six HSP genes were all heat-inducible, of which hsp40 was the most inducible. The temperatures for maximal HSP induction at high and low temperature zone were 36 or 39°C and -3°C, respectively. In the hot zone, all HSP genes have the same initial temperature (33°C) for up-regulation. Low concentrations of Cd(2+) for a short-term promoted the expression of all HSP genes, but not high concentrations or long-term treatments. Cu(2+) stress for 24 h increased expression of nearly all HSP. Four HSP genes changed after starvation. We infer that all six HSP genes are sensitive to heat. This may help understand the absence of P. puparum during the summer and winter. The expression profiles of six HSP genes in P. puparum under heavy metal stress indicates that HSP is a short-term response to cellular distress or injury induced by Cd(2+) and Cu(2+).