分类： 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间： 2016-05-12
Zheng, Yongxiang
Yu, Fei
Wu, Yiming
Si, Longlong
Xu, Huan
Zhang, Chuanling
Xia, Qing
Xiao, Sulong
Wang, Qi
He, Qiuchen
Taira, Kazunari
Zhang, Lihe
Zhou, Demin
Wang, Qi
Chen, Peng
Wang, Jiangyun
摘要： With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure-function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus-host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.
分类： 生物学 >> 生物物理学 >> 生物物理、生物化学与分子生物学 提交时间： 2016-05-11
摘要： Calcium (Ca2+) oscillations play a central role in varieties of cellular processes including fertilization and immune response, but controversy over the regulation mechanisms still exists. It has been known that nitric oxide (NO) dependently regulates Ca2+ signaling in most physiopathological processes. Previous study indicated that eNOS translocation during some pathological process influences intracellular Ca2+ homeostasis. In this study, we investigated the role and mechanism of NO on Ca2+ release by overexpressing eNOS in cytoplasm (Cyto-eNOS) and endoplasmic reticulum (ER-eNOS) of HeLa cells. We found that the properties of Ca2+ release were altered by the overexpression of eNOS. The amplitude and frequency of extracellular ATP (eATP)-induced Ca2+ oscillation were enhanced in both Cyto-eNOS and ER-eNOS cells, respectively. Especially, the enhancement of the amplitude and frequency of the Ca2+ oscillation was much more significant in the ER-eNOS cells than that of Cyto-eNOS cells. Further study indicated that this effect was abrogated by NO inhibitor, 1..-NAME, suggesting it was not an artificial result induced by ER stress. Furthermore, an up-regulated phosphorylation of phospholamban (PLB) was observed and the sarco-endoplasmic reticulum Ca2tATPase (SERCA) function was activated followed by the significant increase in the ER Ca2+ load. Taken together, we revealed a novel regulatory mechanism of Ca2+ oscillation. (C) 2014 Elsevier Inc. All rights reserved.
分类： 生物学 >> 植物学 >> 植物生物化学、植物生物物理学 提交时间： 2016-05-04
摘要： Drought stress has adverse impacts on plant production and productivity. MicroRNAs (miRNAs) are one class of noncoding RNAs regulating gene expression post-transcriptionally. In this study, we employed small RNA and degradome sequencing to systematically investigate the tissue-specific miRNAs responsible to drought stress, which are understudied in tomato. For this purpose, root and upground tissues of two different drought-responsive tomato genotypes (Lycopersicon esculentum as sensitive and L. esculentum var. cerasiforme as tolerant) were subjected to stress with 5% polyethylene glycol for 7days. A total of 699 conserved miRNAs belonging to 578 families were determined and 688 miRNAs were significantly differentially expressed between different treatments, tissues and genotypes. Using degradome sequencing, 44 target genes were identified associated with 36 miRNA families. Drought-related miRNAs and their targets were enriched functionally by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Totally, 53 miRNAs targeted 23 key drought stress- and tissue development-related genes, including DRP (dehydration-responsive protein), GTs (glycosyltransferases), ERF (ethylene responsive factor), PSII (photosystem II) protein, HD-ZIP (homeodomain-leucine zipper), MYB and NAC-domain transcription factors. miR160, miR165, miR166, miR171, miR398, miR408, miR827, miR9472, miR9476 and miR9552 were the key miRNAs functioning in regulation of these genes and involving in tomato response to drought stress. Additionally, plant hormone signal transduction pathway genes were differentially regulated by miR169, miR172, miR393, miR5641, miR5658 and miR7997 in both tissues of both sensitive and tolerant genotypes. These results provide new insight into the regulatory role of miRNAs in drought response with plant hormone signal transduction and drought-tolerant tomato breeding.
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