Screening of binding proteins that interact with human Salvador 1 in a human fetal liver cDNA library by the yeast two-hybrid system

Salvador promotes both cell cycle exit and apoptosis through the modulation of both cyclin E and Drosophila inhibitor of apoptosis protein in Drosophila. However, the cellular function of human Salvador (hSav1) is rarely reported. To screen for novel binding proteins that interact with hSav1, the cDNA of hSav1 was cloned into a bait protein plasmid, and positive clones were screened from a human fetal liver cDNA library by the yeast two-hybrid system. hSav1 mRNA was expressed in yeast and there was no self-activation and toxicity in the yeast strain AH109. Twenty proteins were found to interact with hSav1, including HS1 (haematopoietic cell specific protein1)-associated protein X-1 (HAX-1); neural precursor cell expressed, developmentally down-regulated 9, pyruvate kinase, liver and RBC, cytochrome c oxidase subunit Vb, enoyl coenzyme A hydratase short chain 1, and NADH dehydrogenase (ubiquinone) 1 beta subcomplex, demonstrating that the yeast two-hybrid system is an efficient method for investigating protein interactions. Among the identified proteins, there were many mitochondrial proteins, indicating that hSav1 may play a role in mitochondrial function. We also confirmed the interaction of HAX-1 and hSav1 in mammalian cells. This investigation provides functional clues for further exploration of potential apoptosis-related proteins in disease biotherapy.     

替代宿主增殖松毛虫质型多角体病毒的比较研究

银纹夜蛾幼虫 (Argyrogrammaagnata)对松毛虫CPV(DpCPV)十分敏感 ,本文对两种不同的宿主增殖松毛虫CPV作了比较 ,电镜证实用替代宿主增殖的DpCPV与原毒株多角体 (CPB)和病毒粒子的形态完全一致。 3 %PAGE分析二者的RNA图谱基本一致 ,有大小相同的 2 .98× 10 6- 0 .6 6× 10 6道尔顿的 10条带 ,用它增殖的DpCPV(Aa DpCPV)对松毛虫有相当的毒力 ,每头 3龄幼虫病毒产量平均为 2 .5× 10 8CPB。     

Five New Guaiane Sesquiterpenes from the Endophytic Fungus Xylaria sp. YM 311647 of Azadirachta indica

Five new guaiane sesquiterpenes, 1 – 5 , were isolated from the culture broth of the endophytic fungus Xylaria sp. YM 311647, isolated from Azadirachta indica A. Juss . The structures of these compounds were elucidated on the basis of spectroscopic analyses, and their inhibitory activities against five pathogenic fungi were evaluated. All guaiane sesquiterpenes showed moderate or weak antifungal activities in a broth microdilution assay.     

Endometrial stem cell transplantation restores dopamine production in a Parkinson’s disease model

Parkinson’s disease (PD) is a neurodegenerative disorder caused by the loss of dopaminergic neurons. Adult human endometrial derived stem cells (HEDSC), a readily obtainable type of mesenchymal stem‐like cell, were used to generate dopaminergic cells and for transplantation. Cells expressing CD90, platelet derived growth factor (PDGF)‐Rβ and CD146 but not CD45 or CD31 were differentiated in vitro into dopaminergic neurons that exhibited axon projections, pyramidal cell bodies and dendritic projections that recapitulate synapse formation; these cells also expressed the neural marker nestin and tyrosine hydroxylase, the rate‐limiting enzyme in dopamine synthesis. Whole cell patch clamp recording identified G‐protein coupled inwardly rectifying potassium current 2 channels characteristic of central neurons. A 1‐methyl 4‐phenyl 1,2,3,6‐tetrahydro pyridine induced animal model of PD was used to demonstrate the ability of labelled HEDSC to engraft, migrate to the site of lesion, differentiate in vivo and significantly increase striatal dopamine and dopamine metabolite concentrations. HEDSC are a highly inducible source of allogenic stem cells that rescue dopamine concentrations in an immunocompetent PD mouse model.     

Spred2 Modulates the Erythroid Differentiation Induced by Imatinib in Chronic Myeloid Leukemia Cells

Differentiation induction is currently considered as an alternative strategy for treating chronic myelogenous leukemia (CML). Our previous work has demonstrated that Sprouty-related EVH1 domainprotein2 (Spred2) was involved in imatinib mediated cytotoxicity in CML cells. However, its roles in growth and lineage differentiation of CML cells remain unknown. In this study, we found that CML CD34⁺ cells expressed lower level of Spred2 compared with normal hematopoietic progenitor cells, and adenovirus mediated restoration of Spred2 promoted the erythroid differentiation of CML cells. Imatinib could induce Spred2 expression and enhance erythroid differentiation in K562 cells. However, the imatinib induced erythroid differentiation could be blocked by Spred2 silence using lentiviral vector PLKO.1-shSpred2. Spred2 interference activated phosphorylated-ERK (p-ERK) and inhibited erythroid differentiation, while ERK inhibitor, PD98059, could restore the erythroid differentiation, suggesting Spred2 regulated the erythroid differentiation partly through ERK signaling. Furthermore, Spred2 interference partly restored p-ERK level leading to inhibition of erythroid differentiation in imatinib treated K562 cells. In conclusion, Spred2 was involved in erythroid differentiation of CML cells and participated in imatinib induced erythroid differentiation partly through ERK signaling.     

Response surface methodology to design a selective co-enrichment broth of Escherichia coli, Salmonella spp. and Staphylococcus aureus for simultaneous detection by multiplex PCR

Escherichia coli, Salmonella spp. and Staphylococcus aureus are frequent co-visitors of contaminated foods to cause food-borne diseases. To achieve rapid detection of three organisms by multiplex PCR, a selective co-enrichment broth was considered to design using response surface methodology (RSM) in this work. NaCl, LiCl and KSCN as selective bacterial inhibitors were selected to optimize their concentrations for a matched composition of bacterial biomass with uniform amplification of three targets. Central composite design was employed to collect the data and fit the responses. Three quadratic polynomial models were derived by computer simulation. A statistical analysis was carried out to explore the effects of the variables on the composition of bacterial biomass and PCR amplification yields. In the end, a novel broth (ESS-3 broth) of NaCl 1.60%, LiCl 0.70%, KSCN 0.10% was formulated to allow co-enrichment of the target pathogens and suppress growth of some non-target pathogens. The simultaneous detection of E. coli, Salmonella spp. and S. aureus was developed on a rapid, convenient and sensitive method consisting of selective co-enrichment in ESS-3 broth, DNA extraction with the boiling method and robust test by multiplex PCR. Our work provided broader application of RSM for the simultaneous detection of other combinations of multiple pathogens.     

REFOLDdb: a new and sustainable gateway to experimental protocols for protein refolding

Background

More than 7000 papers related to “protein refolding” have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource – “REFOLDdb” that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest.

Results

We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17^th, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/.

Conclusion

REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.

     

Disomic Thinopyrum intermedium addition lines in wheat with barley yellow dwarf virus resistance and with rust resistances.

Zhong 5 is a partial amphiploid (2n = 56) between Triticum aestivum (2n = 42) and Thinopyrum intermedium (2n = 42) carrying all the chromosomes of wheat and seven pairs of chromosomes from Th. intermedium. Following further backcrossing to wheat, six independent stable 2n = 44 lines were obtained representing 4 disomic chromosome addition lines. One chromosome confers barley yellow dwarf virus (BYDV) resistance, whereas two other chromosomes carry leaf and stem rust resistance; one of the latter also confers stripe rust resistance. Using RFLP and isozyme markers we have shown that the extra chromosome in the Zhong 5-derived BYDV resistant disomic addition lines (Z1, Z2, or Z6) belongs to the homoeologous group 2. It therefore carries a different locus to the BYDV resistant group 7 addition, L1, described previously. The leaf, stem, and stripe rust resistant line (Z4) carries an added group 7 chromosome. The line Z3 has neither BYDV nor rust resistance, is not a group 2 or group 7 addition, and is probably a group 1 addition. The line Z5 is leaf and stem rust resistant, is not stripe rust resistant, and its homoeology remains unknown.