蒙古黄芪病程相关蛋白AmPR-10于大肠杆菌不同载体的可溶性表达策略研究

蒙古黄芪病程相关蛋白（Astragalus membranaceus pathogenesis-related protein-10, AmPR-10）具有核酸酶活性,对黄芪生长发育及抗病机制具有重要意义。但从天然黄芪中提取蛋白质的传统方法成本较高,蛋白得率较少。研究首次利用大肠杆菌表达体系对AmPR-10进行可溶性外源表达,构建了3种重组子：①以pET28a为载体构建pET28a-AmPR-10;②以pET30a为载体构建pET30a-AmPR-10;③以pET30a为载体、大肠杆菌分子伴侣skp修饰构建pET30a-skp-AmPR-10。通过SDS-PAGE分析,目的蛋白可溶性表达量比较结果是：pET30a-skp-AmPR-10 > pET30a-AmPR-10 > pET28a-AmPR-10。由蛋白质三级结构模拟分析发现,载体pET-30a上的标签S-tag通过影响AmPR-10局部α螺旋构象而提高目标蛋白的可溶性表达,分子伴侣skp通过提高融合蛋白整体α螺旋比例进一步提升目标蛋白可溶性表达量。研究解决了目前从天然黄芪中提取蛋白操作复杂、提取量小的问题。同时,经测定,目的蛋白具有核酸酶活性,为外源AmPR-10相关活性研究提供了依据。     

The Relationship Between Insulin Resistance and CpG Island Methylation of LMNA Gene in Polycystic Ovary Syndrome

We sought to investigate the relationship between the changes of CpG island methylation status of LMNA gene and insulin resistance in polycystic ovary syndrome (PCOS) patients. The genome-wide methylation microarray screening was done in three PCOS cases of insulin resistance and one case of a normal woman. The PCOS insulin resistance-related genes were identified as indicated by the results of gene chip screening. Then, 24 cases of insulin-resistant PCOS patients and 24 cases of normal individuals were studied to identify the effects of the candidate genes using genome-wide study of DNA from the peripheral blood analyzed by MassARRAY^®EpiTYPER? DNA methylation analysis technique. We found that the methylation status of CpG island in the promoter area of LMNA gene was changed. The 20 CG sites in CpG island of LMNA gene were examined using case control experiment among which 12 CpG sites differed significantly (P < 0.05) between two groups while the remaining eight CpG sites differed non-significantly. We, therefore, concluded that the changes in the hypermethylation status of CpG island of LMNA gene were related to the insulin resistance in PCOS patients, indicating that this gene may be involved in the regulation of PCOS-associated insulin resistance.     

Cloning of the crustacean hyperglycemic hormone and evidence for molt-inhibiting hormone within the central nervous system of the blue crab Portunus pelagicus

The crustacean X-organ–sinus gland (XO–SG) complex controls molt-inhibiting hormone (MIH) production, although extra expression sites for MIH have been postulated. Therefore, to explore the expression of MIH and distinguish between the crustacean hyperglycemic hormone (CHH) superfamily, and MIH immunoreactive sites (ir) in the central nervous system (CNS), we cloned a CHH gene sequence for the crab Portunus pelagicus (Ppel-CHH), and compared it with crab CHH-type I and II peptides. Employing multiple sequence alignments and phylogenic analysis, the mature Ppel-CHH peptide exhibited residues common to both CHH-type I and II peptides, and a high degree of identity to the type-I group, but little homology between Ppel-CHH and Ppel-MIH (a type II peptide). This sequence identification then allowed for the use of MIH antisera to further confirm the identity and existence of a MIH-ir 9 kDa protein in all neural organs tested by Western blotting, and through immunohistochemistry, MIH-ir in the XO, optic nerve, neuronal cluster 17 of the supraesophageal ganglion, the ventral nerve cord, and cell cluster 22 of the thoracic ganglion. The presence of MIH protein within such a diversity of sites in the CNS, and external to the XO–SG, raises new questions concerning the established mode of MIH action.     

Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. Expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. Tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.     

VRAA: virtualized resource auction and allocation based on incentive and penalty

Virtualization is widely used in cloud computing environments to efficiently manage resources, but it also raises several challenges. One of them is the fairness issue of resource allocation among virtual machines. Traditional virtualized resource allocation approaches distribute physical resources equally without taking into account the actual workload of each virtual machine and thus often leads to wasting. In this paper, we propose a virtualized resource auction and allocation model (VRAA) based on incentive and penalty to correct this wasting problem. In our approach, we use Nash equilibrium of cooperative games to fairly allocate resources among multiple virtual machines to maximize revenue of the system. To illustrate the effectiveness of the proposed approach, we then apply the basic laws of auction gaming to investigate how CPU allocation and contention can affect applications’ performance (i.e., response time), and its effect on CPU utilization. We find that in our VRAA model, the fairness index is high, and the resource allocation is closely proportional to the actual workloads of the virtual machines, so the wasting of resources is reduced. Experiment results show that our model is general, and can be applied to other virtualized non-CPU resources.     

Inhibition of in vivo Slicer activity of Argonaute protein 1 by the viral 2b protein independent of its dsRNA‐binding function

The 2b protein of Cucumber mosaic virus (CMV) has several unique properties, such as targeting to the nucleolus and interaction with both Argonautes (AGOs) and short and long double‐stranded RNA (dsRNA). We have recently uncoupled the domain requirements for dsRNA binding and nucleolar targeting from the physical interactions with AGO proteins, and have found that the direct 2b–AGO interaction is sufficient to inhibit the in vitro AGO1 Slicer function independent of the other biochemical properties of 2b. Because the AGO binding activity of 2b is not required for its suppressor function in vivo, this raises the question of whether in vivo 2b–AGO interaction is possible to inhibit the in vivo AGO Slicer function. In this study, by taking advantage of a technology for the production of artificial trans‐acting small interfering RNA (tasiRNA), a process uniquely associated with AGO1‐mediated in vivo Slicer activity, we demonstrated that the expression of the 2b protein in planta interfered with the production of tasiRNA. Through further detailed analysis with deletion mutants of 2b proteins, we found that the inhibition of in vivo AGO1 Slicer function required the nucleolar localization signal (NoLS), in addition to the AGO‐binding domain, of the 2b protein. Our finding demonstrates that in vivo 2b–AGO1 interaction is sufficient to inhibit AGO1 Slicer function independent of the dsRNA‐binding activity of the 2b protein.     

Biochemical Characterization of a First Fungal Esterase from Rhizomucor miehei Showing High Efficiency of Ester Synthesis

Background

Esterases with excellent merits suitable for commercial use in ester production field are still insufficient. The aim of this research is to advance our understanding by seeking for more unusual esterases and revealing their characterizations for ester synthesis.

Methodology/Principal Findings

A novel esterase-encoding gene from Rhizomucor miehei (RmEstA) was cloned and expressed in Escherichia coli. Sequence analysis revealed a 975-bp ORF encoding a 324-amino-acid polypeptide belonging to the hormone-sensitive lipase (HSL) family IV and showing highest similarity (44%) to the Paenibacillus mucilaginosus esterase/lipase. Recombinant RmEstA was purified to homogeneity: it was 34 kDa by SDS-PAGE and showed optimal pH and temperature of 6.5 and 45°C, respectively. The enzyme was stable to 50°C, under a broad pH range (5.0–10.6). RmEstA exhibited broad substrate specificity toward p-nitrophenol esters and short-acyl-chain triglycerols, with highest activities (1,480 U mg⁻¹ and 228 U mg⁻¹) for p-nitrophenyl hexanoate and tributyrin, respectively. RmEstA efficiently synthesized butyl butyrate (92% conversion yield) when immobilized on AOT-based organogel.

Conclusion

RmEstA has great potential for industrial applications. RmEstA is the first reported esterase from Rhizomucor miehei.