SSRs isolated from apple can identify polymorphism and genetic diversity in pear

Apple simple sequence repeats (SSRs) were intergenerically applied to the characterization of 36 pear accessions, including 19 Japanese pears (Pyrus pyrifolia), 7 Chinese pears (P. bretschneideri, P. ussuriensis), 5 European pears (P. communis), 3 wild relatives (P. calleryana), and 2 hybrids between P. pyrifolia and P. communis. All of the tested SSR primers derived from apple produced discrete amplified fragments in all pear accessions. Nucleotide repeats were detected in the amplified bands by both Southern blot and sequencing analysis, and nucleotide sequences of pear were compared with those of apple. The differences in fragment size among pear or between pear and apple were, in many cases, due to the differences in repeat number. Interestingly, the DNA sequence of flanking regions in apple was highly conserved in pear. Hybrids from P. pyrifolia×P. communis showed one fragment inherited from each parent in all scorable cases, which suggested that each primer pair amplified fragments originating from the same locus. A total of 79 alleles were detected from seven SSR loci in pear, and all pear varieties except for the mutants could be differentiated. In conclusion, SSRs isolated from apple are highly conserved in pear and could be utilized as DNA markers in the latter genus. Received: 17 July 2000 / Accepted: 22 September 2000     

Development of microsatellite markers in the Japanese pear (Pyrus pyrifolia Nakai)

Thirteen polymorphic microsatellite loci were developed in the Japanese pear (Pyrus pyrifolia Nakai) by using an enriched genomic library. The obtained microsatellite loci showed a high degree of polymorphism in the Japanese pear with 3–6 alleles per locus. The average values of observed and expected heterozygosities among these 13 loci were 0.69 and 0.71, respectively. Ten microsatellites could successfully amplify loci in the European pear (Pyrus communis L.), which were highly polymorphic as well.     

In vitro binding of an orally active platinum antitumor drug, JM216 to metallothionein

The in vitro binding of an orally active anticancer drug JM216 to metallothionein is firstly investigated in this paper. It is revealed that a redox reaction following a substitution reaction from JM216 with rabbit liver Zn₇MT-II is presented. The reaction feature, the metal binding stoichiometry and the oxidation states of platinum and sulfur in the products are studied by UV-visible, chromatography and X-ray photoelectron spectroscopy methods. Parts of MT are oxidized to precipitate products with intra and intermolecular CyS-SCy disulfides linkages. Pt(IV) is reduced to its Pt(II) counterpart. And the reduced Pt(II) replace the metal ions in native MTs. Meanwhile it can also cause the dimerization of MT. Increasing the reaction ratio of JM216 to MT leads to a concomitant increase in the apparent yield of the precipitate and dimeric products and the elevation of the binding stoichiometry of Pt to the protein. Based on the experimental data, the reaction mechanism between JM216 and Zn₇MT-II in vitro are discussed.     

Mono-(2-Ethylhexyl) Phthalate Induces Injury in Human Umbilical Vein Endothelial Cells

Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate (DEHP), is a widespread environmental contaminant and has been proved to have potential adverse effects on the reproductive system, carcinogenicity, liver, kidney and developmental toxicities. However, the effect of MEHP on vascular system remains unclear. The main purpose of this study was to evaluate the cytotoxic effects of MEHP on human umbilical endothelial cells (HUVEC) and its possible molecular mechanism. HUVEC cells were treated with MEHP (0, 6.25, 12.5, 25,50 and 100 µM), and the cellular apoptosis and mitochondrial membrane potential as well as intracellular reactive oxygen species were determined. In present study, MEHP induced a dose-dependent cell injury in HUVEC cell via an apoptosis pathway as characterized by increased percentage of sub-G1, activation of caspase-3, -8and -9, and increased ratio of Bax/bcl-2 mRNA and protein expression as well as cytochrome C releasing. In addition, there was obvious oxidative stress, represented by decreased glutathione level, increased malondialdehyde level and superoxide dismutase activity. N-Acetylcysteine, as an antioxidant that is a direct reactive oxygen species scavenger, could effectively block MEHP-induced reactive oxygen species generation, mitochondrial membrane potential loss and cell apoptosis. These data indicated that MEHP induced apoptosis in HUVEC cells through a reactive oxygen species-mediated mitochondria-dependent pathway.     

Expression Patterns and Potential Biological Roles of Dip2a

Disconnected (disco)-interacting protein 2 homolog A is a member of the DIP2 protein family encoded by Dip2a gene. Dip2a expression pattern has never been systematically studied. Functions of Dip2a in embryonic development and adult are not known. To investigate Dip2a gene expression and function in embryo and adult, a Dip2a-LacZ mouse model was generated by insertion of β-Gal cDNA after Dip2a promoter using CRISPR/Cas9 technology. Dip2a-LacZ mouse was designed to be a lacZ reporter mouse as well as a Dip2a knockout mouse. Heterozygous mice were used to study endogenous Dip2a expression and homozygotes to study DIP2A-associated structure and function. LacZ staining indicated that Dip2a is broadly expressed in neuronal, reproductive and vascular tissues, as well as in heart, kidney, liver and lung. Results demonstrate that Dip2a is expressed in ectoderm-derived tissues in developing embryos. Adult tissues showed rich staining in neurons, mesenchymal, endothelial, smooth muscle cells and cardiomyocytes by cell types. The expression pattern highly overlaps with FSTL1 and supports previous report that DIP2A to be potential receptor of FSTL1 and its protective roles of cardiomyocytes. Broad and intense embryonic and adult expression of Dip2a has implied their multiple structural and physiological roles.     

circRNAs在植物中的研究进展

circular RNA(circRNA)是一类具有闭合环状结构的内源性非编码RNA,广泛存在于多种真核生物中,具有结构稳定、序列保守、表达特异性等特征。研究表明circRNAs可作为海绵(sponge)吸附microRNA(miRNA)并参与其表达调控过程,也可通过与蛋白互作调控基因表达等生物过程;发现circRNAs不仅参与植物激素信号转导等生理过程,而且还能在植物响应逆境胁迫中起到重要作用。该文主要对近年来国内外有关circRNAs的类型、形成机制、功能及其在植物生长发育过程中的研究进展进行了综述,并讨论了circRNAs的研究意义及存在的问题,为进一步研究circRNAs在植物中的作用机制及其基因调控网络提供参考。     

A highly glucose-tolerant GH1 β-glucosidase with greater conversion rate of soybean isoflavones in monogastric animals

In the feed industry, β-glucosidase has been widely used in the conversion of inactive and bounded soybean isoflavones into active aglycones. However, the conversion is frequently inhibited by the high concentration of intestinal glucose in monogastric animals. In this study, a GH1 β-glucosidase (AsBG1) with high specific activity, thermostability and glucose tolerance (IC₅₀ = 800 mM) was identified. It showed great glucose tolerance against substrates with hydrophobic aryl ligands (such as pNPG and soy isoflavones). Using soybean meal as the substrate, AsBG1 exhibited higher hydrolysis efficiency than the GH3 counterpart Bgl3A with or without the presence of glucose in the reaction system. Furthermore, it is the first time to find that the endogenous β-glucosidase of soybean meal, mostly belonging to GH3, plays a role in the hydrolysis of soybean isoflavones and is highly sensitive to glucose. These findings lead to a conclusion that the GH1 rather than GH3 β-glucosidase has prosperous application advantages in the conversion of soybean isoflavones in the feed industry.

     

Classical swine fever virus non-structural protein 4B binds tank-binding kinase 1

Classical swine fever (CSF) is a contagious disease with a high mortality rate and is caused by classical swine fever virus (CSFV). CSFV non-structural protein 4B (NS4B) plays a crucial role in CSFV replication and pathogenicity. However, precisely how NS4B exerts these functions remains unknown, especially as there are no reports relating to potential cellular partners of CSFV NS4B. Here, a yeast two-hybrid (Y2H) system was used to screen the cellular proteins interacting with NS4B from a porcine alveolar macrophage (PAM) cDNA library. The protein screen along with alignment using the NCBI database revealed 14 cellular proteins that interact with NS4B: DDX39B, COX7C, FTH1, MAVS, NR2F6, RPLP1, PSMC4, FGL2, MKRN1, RPL15, RPS3, RAB22A, TP53BP2 and TBK1. These proteins mostly relate to oxidoreductase activity, signal transduction, localization, biological regulation, catalytic activity, transport and metabolism by GO categories. Tank-binding kinase 1 (TBK1) was chosen for further confirmation. The NS4B-TBK1 interaction was further confirmed by subcellular co-location, co-immunoprecipitation and glutathione S-transferase pull-down assays. This study offers a theoretical foundation for further understanding of the diversity of NS4B functions in relation to viral infection and subsequent pathogenesis.