Affinity chromatographic purification of human immunoglobulin a from chinese hamster ovary cell culture supernatant

Hexamer peptide ligand HWRGWV, initially screened from a solid phase combinatorial peptide library for immunoglobulins G (IgG) purification, is shown to also have potential for immunoglobulin A (IgA) purification. The determined dissociation constants for hIgA on HWRGWV resins at three different peptide densities from 0.11 to 0.55 meq/g fall in the range of 10^?6–10^?7 M, which are somewhat lower than those for hIgG. Although relatively low dynamic binding capacity (DBC) in the range of 9.2–16.8 mg IgA/mL resin at linear flow rates from 173 to 35 cm/h were obtained for IgA compared to IgG, the DBC value of HWRGWV for IgA is much greater than current commercially available affinity ligands. Although relatively lower binding affinity to secretory IgA compared to monomeric IgA was observed, the peptide ligand resins exhibit great potential for large‐scale purification of both human IgA and secretory IgA. Recoveries of 96.0% and 94.3%, and purities of 90.3% and 91.7% were achieved for human IgA and secretory IgA purification, respectively, from spiked Chinese hamster ovary cell culture supernatants without an extra afterwash step. Over 95% in purities were achieved for IgA and secretory IgA with an extra afterwash step; however, the recoveries would decrease at least 15% and 40% for IgA and secretory IgA, respectively. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

CD106 Identifies a Subpopulation of Mesenchymal Stem Cells with Unique Immunomodulatory Properties

Mesenchymal stem cells (MSCs) reside in almost all of the body tissues, where they undergo self-renewal and multi-lineage differentiation. MSCs derived from different tissues share many similarities but also show some differences in term of biological properties. We aim to search for significant differences among various sources of MSCs and to explore their implications in physiopathology and clinical translation. We compared the phenotype and biological properties among different MSCs isolated from human term placental chorionic villi (CV), umbilical cord (UC), adult bone marrow (BM) and adipose (AD). We found that CD106 (VCAM-1) was expressed highest on the CV-MSCs, moderately on BM-MSCs, lightly on UC-MSCs and absent on AD-MSCs. CV-MSCs also showed unique immune-associated gene expression and immunomodulation. We thus separated CD106⁺cells and CD106⁻cells from CV-MSCs and compared their biological activities. Both two subpopulations were capable of osteogenic and adipogenic differentiation while CD106⁺CV-MSCs were more effective to modulate T helper subsets but possessed decreased colony formation capacity. In addition, CD106⁺CV-MSCs expressed more cytokines than CD106⁻CV-MSCs. These data demonstrate that CD106 identifies a subpopulation of CV-MSCs with unique immunoregulatory activity and reveal a previously unrecognized mechanism underlying immunomodulation of MSCs.     

CSTP1, a Novel Protein Phosphatase,Blocks Cell Cycle,Promotes Cell Apoptosis,and Suppresses Tumor Growth of Bladder Cancer by Directly Dephosphorylating Akt at Ser473 Site

Akt/protein kinase B is a pivotal component downstream of phosphatidylinositol 3-kinase (PI3K) pathway, whose activity regulates the balance between cell survival and apoptosis. Phosphorylation of Akt occurs at two key sites either at Thr308 site in the activation loop or at Ser473 site in the hydrophobic motif. The phosphorylated form of Akt (pAkt) is activated to promote cell survival. The mechanisms of pAkt dephosphorylation and how the signal transduction of Akt pathway is terminated are still largely unknown. In this study, we identified a novel protein phosphatase CSTP1(complete s transactivated protein 1), which interacts and dephosphorylates Akt specifically at Ser473 site in vivo and in vitro, blocks cell cycle progression and promotes cell apoptosis. The effects of CSTP1 on cell survival and cell cycle were abrogated by depletion of phosphatase domain of CSTP1 or by expression of a constitutively active form of Akt (S473D), suggesting Ser473 site of Akt as a primary cellular target of CSTP1. Expression profile analysis showed that CSTP1 expression is selectively down-regulated in non-invasive bladder cancer tissues and over-expression of CSTP1 suppressed the size of tumors in nude mice. Kaplan-Meier curves revealed that decreased expression of CSTP1 implicated significantly reduced recurrence-free survival in patients suffered from non-invasive bladder cancers.     

Genome-Wide Identification,Characterization, and Stress-Responsive Expression Profiling of Genes Encoding LEA (Late Embryogenesis Abundant) Proteins in Moso Bamboo (Phyllostachys edulis)

Late embryogenesis abundant (LEA) proteins have been identified in a wide range of organisms and are believed to play a role in the adaptation of plants to stress conditions. In this study, we performed genome-wide identification of LEA proteins and their coding genes in Moso bamboo (Phyllostachys edulis) of Poaceae. A total of 23 genes encoding LEA proteins (PeLEAs) were found in P. edulis that could be classified to six groups based on Pfam protein family and homologous analysis. Further in silico analyses of the structures, gene amount, and biochemical characteristics were conducted and compared with those of O. sativa (OsLEAs), B. distachyon (BdLEAs), Z. mays (ZmLEAs), S. bicolor (SbLEAs), Arabidopsis, and Populus trichocarpa. The less number of PeLEAs was found. Evolutionary analysis revealed orthologous relationship and colinearity between P. edulis, O. sativa, B. distachyon, Z. mays, and S. bicolor. Analyses of the non-synonymous (Ka) and synonymous (Ks)substitution rates and their ratios indicated that the duplication of PeLEAs may have occurred around 18.8 million years ago (MYA), and divergence time of LEA family among the P. edulis-O. sativa and P. edulis–B. distachyon, P. edulis-S. bicolor, and P. edulis-Z. mays was approximately 30 MYA, 36 MYA, 48 MYA, and 53 MYA, respectively. Almost all PeLEAs contain ABA- and (or) stress-responsive regulatory elements. Further RNA-seq analysis revealed approximately 78% of PeLEAs could be up-regulated by dehydration and cold stresses. The present study makes insights into the LEA family in P. edulis and provides inventory of stress-responsive genes for further functional validation and transgenic research aiming to plant genetic improvement of abiotic stress tolerance.     

苹果树腐烂病菌非核糖体多肽合成酶基因VmNRPS12的功能

【目的】非核糖体多肽合成酶(NRPS)在植物病原真菌与其寄主互作过程中发挥着重要作用,明确Vm NRPS12基因在苹果树腐烂病菌致病过程中的功能,将为今后深入研究苹果树腐烂病菌NRPS作用机制提供理论依据。【方法】基于苹果树腐烂病菌全基因组数据,得到VmNRPS12基因。运用qRT-PCR技术分析VmNRPS12在侵染初期的表达水平,利用Double-joint PCR和PEG介导的原生质体转化获得该基因抗潮霉素的突变体,对突变体进行PCR检测及Southern blot验证得到敲除突变体,进一步通过重新导入该基因全长片段获得互补突变体,最后对野生型、敲除突变体和互补突变体进行菌落、产孢及致病力观察,对检测数据用SPSS软件进行差异显著性分析。【结果】定量分析显示该基因在侵染初期显著上调表达,且接种48 h后的表达量是对照的138.6倍。该基因的敲除突变体在营养生长及产孢方面与野生型菌株03-8相比无显著性差异,但致病力与野生型菌株03-8相比显著减弱,且互补突变体致病力近似恢复至野生型水平。【结论】VmNRPS12基因与苹果树腐烂病菌致病性相关。     

特定环状RNA表达与衰老的关系研究*

目的:研究小鼠不同组织器官中环状RNA的表达量在年老与年幼中的差异,揭示环状RNA与衰老的联系。方法:通过对不同组织器官的总RNA的提取,分别逆转录,并扩增circUSP34、circTCF4、circTCF20、circZFP64、circPWWP2A、circLIN54,通过凝胶电泳比较其在年轻小鼠与年老小鼠的组织器官中的表达情况,明确上述环状RNA与衰老的联系。结果:环状RNA在不同年龄小鼠的组织器官中存在广泛性的差异性表达,不同组织不同环状RNA有着不同的表达量的改变,其与衰老存在联系。在卵巢中除circTCF4,其余五种环状RNA表达量均与年龄负相关;在肾脏中,除circTCF4与circPWWP2A,其余与年龄呈负相关;在乳腺中只有circUSP3与衰老呈负相关;而在肠道与生长相关的只有上述后三种环状RNA,均呈正相关。其他样本与组织的发育阶段有不同的相关性,具体在结果体现。结论:环状RNA的表达量在组织中与年龄的增大有着显著相关性,环状RNA可能广泛地参与了不同的衰老通路,起到不同的生物学作用,其在衰老的产生中扮演重要角色。