The cellular response to DNA damage induced by the enediynes C-1027 and neocarzinostatin includes hyperphosphorylation and increased nuclear retention of replication protein a (RPA) and trans inhibition of DNA replication

This study examined the cellular response to DNA damage induced by antitumor enediynes C-1027 and neocarzinostatin. Treatment of cells with either agent induced hyperphosphorylation of RPA32, the middle subunit of replication protein A, and increased nuclear retention of RPA. Nearly all of the RPA32 that was not readily extractable from the nucleus was hyperphosphorylated, compared to < or =50% of the soluble RPA. Enediyne concentrations that induced RPA32 hyperphosphorylation also decreased cell-free SV40 DNA replication competence in extracts of treated cells. This decrease did not result from damage to the DNA template, indicating trans-acting inhibition of DNA replication. Enediyne-induced RPA hyperphosphorylation was unaffected by the replication elongation inhibitor aphidicolin, suggesting that the cellular response to enediyne DNA damage was not dependent on elongation of replicating DNA. Neither recovery of replication competence nor reversal of RPA effects occurred when treated cells were further incubated in the absence of drug. C-1027 and neocarzinostatin doses that caused similar levels of DNA damage resulted in equivalent increases in RPA32 hyperphosphorylation and RPA nuclear retention and decreases in replication activity, suggesting a common response to enediyne-induced DNA damage. By contrast, DNA damage induced by C-1027 was at least 5-fold more cytotoxic than that induced by neocarzinostatin.     

家蚕let-7 microRNA靶基因Bmlin-41的克隆表达与调控

异时性基因调控细胞增殖和个体发育阶段的转换。家蚕异时性基因在家蚕变态发育过程中也很可能具有重要的调控作用,但它们的表达模式、生物学功能以及与micro RNA之间的关系却鲜有报道。本研究首先利用果蝇同源基因lin-41搜索家蚕基因组数据库中相似序列,设计引物扩增Bmlin-41的编码序列,克隆了家蚕Bmlin-41基因CDS,其长度为2 166 bp,编码721个氨基酸,含有B-box和NHL结构域;随后,利用RT-PCR、q PCR技术并结合已有的家蚕全基因组芯片数据研究了Bmlin-41在家蚕中的时空表达模式,发现Bmlin-41在从家蚕胚胎到成虫的发育过程中呈逐渐递增的表达趋势,在五龄3 d不同组织中,于卵巢里表达量最高,精巢和中肠次之,而其余组织中低量表达或不表达;最后,利用3′RACE克隆了Bmlin-41基因的3′UTR,全长1 434 bp,用在线软件RNAhybrid预测发现Bmlin-41的3′UTR上存在bmo-let-7靶位点,构建了含Bmlin-41 3′UTR的双荧光素酶报告基因载体,在S2细胞上共转染Bmlin-41 3′UTR和bmo-let-7的模拟物(Mimics)和拮抗剂(Antagomir),bmo-let-7 mimics显著下调Bmlin-41,bmo-let-7 antagomir显著上调Bmlin-41,证实了Bmlin-41是bmo-let-7的靶基因。以上研究结果为深入研究let-7 mi RNA和Bmlin-41的功能,揭示Bmlin-41和bmo-let-7在家蚕变态发育过程中的调控关系提供了新的线索。     

Genetic Diversity and Population Structure Patterns in Chinese Cherry (<Emphasis Type="Italic">Prunus pseudocerasus</Emphasis> Lindl) Landraces

Chinese cherry (Prunus pseudocerasus Lindl.) is an ancient fruit crop with highly economic and ornamental values. It originated in China and the cultivation history can be traced back to 3,000 - 4,000 years ago. Over such a long-term domestication process, a large number of genetic variations have been accumulated in different landraces. However, their utilization for cultivar improvement is limited by the scarcity of information involving genetic diversity and population structure. Here, 17 populations comprised of 140 individuals were collected from four geographic areas: Sichuan Basin (SC), Qinglin Mountain (QL), Yungui Plateau (YG) and North of China (NC), and analyzed using a set of 20 microsatellite markers. In total, 126 polymorphic loci were generated, with 6.3 loci per primer. The global expected heterozygosity (He = 0.63) and Shannon information index (I = 1.23) implied a moderately high level of genetic variation. Two major clusters (cluster 1 and cluster 2) were demonstrated based on population structure analysis, which implied the presence of two potential domestication sites of Chinese cherry landraces. Individuals from SC were assigned to cluster 1 and those from QL, YG and NC were grouped into cluster 2. Samples from QL region contained the most plentiful admixture genetic components, implied the possibility of being one transition region of genetic variation. Moreover, botanical characteristics, such as long lifespan, inbreeding preference as well as vegetative propagation, might lead to a relatively low level but significant genetic divergence among populations. Finally, conservation strategies were proposed to protect these valuable natural germplasm based on these results.     

Bioaugmentation accelerates the shift of bacterial community structure against shock load: a case study of coking wastewater treatment by zeolite-sequencing batch reactor

Bioaugmentation with degrading bacteria is an effective method to improve the treatment of refractory industrial wastewater; nevertheless there were controversial opinions about the fate of inoculated bacteria and microbial community dynamics. In this study, two lab-scale sequencing batch reactors filled with modified zeolite were used to treat a coking wastewater with pyridine and quinoline shock load, and a bacterial consortium containing three degrading strains was added in one reactor for bioaugmentation. During 120-day operation, the bioaugmented reactor removed over 99 % pyridine, 99 % quinoline, 85 % TOC, 65 % COD, and 95 % NO₃ ^?-N with higher resistance to the shock load than the non-bioaugmented reactor. Based on the terminal restriction fragment length polymorphism of 16S rDNA, bacterial community diversity increased in the bioaugmented reactor. Principal component analysis revealed that, to cope with the shock load, the indigenous bacterial community recovered to the initial structure by acclimatizing itself constantly to the inhospitable environment; but bioaugmentation accelerated the shift of whole bacterial community, resulting in a far different structure from the initial one. Canonical correspondence analysis indicated that the environmental parameters of pyridine, quinoline, TOC, and NO₃ ^?-N had close negative correlations with bioaugmentation; and NH₃-N and COD were the main parameters to impact on the bacterial community changes and treatment efficiency.     

Effects of Nickel Chloride on the Erythrocytes and Erythrocyte Immune Adherence Function in Broilers

This study was conducted to investigate the immune adherence function of erythrocytes and erythrocyte induced by dietary nickel chloride (NiCl₂) in broilers fed on a control diet and three experimental diets supplemented with 300, 600, and 900 mg/kg NiCl₂ for 42 days. Blood samples were collected from five broilers in each group at 14, 28, and 42 days of age. Changes of erythrocyte parameters showed that total erythrocyte count (TEC), hemoglobin (Hb) contents, and packed cell volume (PCV) were significantly lower (p?p?p?p?+/K⁺-ATPase) and calcium adenosine triphosphatase (Ca²⁺-ATPase) activities were significantly decreased (p?p?2-treated groups. The results of erythrocyte immune adherence function indicated that erythrocyte C_3b receptor rosette rate (E-C_3bRR) was significantly decreased (p?p?p?p?2 in excess of 300 mg/kg caused anemia and impaired the erythrocytic integrity, erythrocytic ability to transport oxygen, and erythrocyte immune adherence function in broilers. Impairment of the erythrocytes and erythrocyte immune adherence function was one of main effect mechanisms of NiCl₂ on the blood function.     

Overexpression of Camellia sinensis H1 histone gene confers abiotic stress tolerance in transgenic tobacco

Key message

Overexpression of CsHis in tobacco promoted chromatin condensation, but did not affect the phenotype. It also conferred tolerance to low-temperature, high-salinity, ABA, drought and oxidative stress in transgenic tobacco.

Abstract

H1 histone, as a major structural protein of higher-order chromatin, is associated with stress responses in plants. Here, we describe the functions of the Camellia sinensis H1 Histone gene (CsHis) to illustrate its roles in plant responses to stresses. Subcellular localization and prokaryotic expression assays showed that the CsHis protein is localized in the nucleus, and its molecular size is approximately 22.5 kD. The expression levels of CsHis in C. sinensis leaves under various conditions were investigated by qRT-PCR, and the results indicated that CsHis was strongly induced by various abiotic stresses such as low-temperature, high-salinity, ABA, drought and oxidative stress. Overexpression of CsHis in tobacco (Nicotiana tabacum) promoted chromatin condensation, while there were almost no changes in the growth and development of transgenic tobacco plants. Phylogenetic analysis showed that CsHis belongs to the H1C and H1D variants of H1 histones, which are stress-induced variants and not the key variants required for growth and development. Stress tolerance analysis indicated that the transgenic tobacco plants exhibited higher tolerance than the WT plants upon exposure to various abiotic stresses; the transgenic plants displayed reduced wilting and senescence and exhibited greater net photosynthetic rate (Pn), stomatal conductance (Gs) and maximal photochemical efficiency (Fv/Fm) values. All the above results suggest that CsHis is a stress-induced gene and that its overexpression improves the tolerance to various abiotic stresses in the transgenic tobacco plants, possibly through the maintenance of photosynthetic efficiency.