Identification of amino compounds synthesized and translocated in symbiotic Parasponia

We investigated the synthesis and translocation of amino compounds in Parasponia, a genus of the Ulmaceae that represents the only non-legumes known to form a root nodule symbiosis with rhizohia. In the xylem sap of P. andersonii we identified asparagine. aspartate. glutamine, glutamated significant quantities of a non-protein amino acid. 4-methylglutamte(2-amino-4-methylpentanedioic acid). This identification was confirmed by two methods, capillary gas chromatography (GC) electron ionization (El) mass spectrometry (MS) and reverse phase high pressure liquid chromatography (HPLC) analysis of derivatized compounds. In leaf, root and nodule samples from P. andersonii and P. parviflora we also identified the related compounds 4-methyleneglutamate and 4-methyleneglulamine. Using ¹⁵N₂ labelling and GC-Ms analysis of root nodule extracts we followed N₂ fixation and ammonia assimilation in P. andersonii root nodules and observed Label initially in glutamine and subsequently in glutamate, suggesting operation of the glutamine synthetase/glutamine:2-oxoglutarate aminotransferase (GS/GOGAT) pathway. Importantly, we observed the incorporation of significant quantities of ¹⁵N into 4-methylglutamate in nodules, demonstrating the de nova synthesis of this non protein amino acid and suggesting a role in the translation of N in symbioticParasponia.     

Growth responses of rice in ammonium-based nutrient solution with variable calcium supply

It is commonly known that calcium promotes NO₃ ^- uptake in many crop species. However, calcium enhancement of NH₄ ⁺ uptake by plants has received little attention. This study aimed to evaluate the effect of Ca supplements on NH₄ ⁺ uptake and plant growth in solution cultured rice. Supplemental Ca applied at vegetative and reproductive phases of plant ontogeny tended to stimulate NH₄ ⁺ absorption, and accordingly resulted in a better straw and grain yield. However, excessively supplied Ca (400 ppm) was detrimental to plant growth. Increases in straw and grain yield observed at Ca levels up to 300 ppm were linked to the Ca-enhanced activities of glutamine synthetase (GS), glutamate synthase (GOGAT), and ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco).     

GPAT2, a mitochondrial outer membrane protein,in piRNA biogenesis in germline stem cells

piRNA (PIWI-interacting RNA) is a germ cell–specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.     

Development of a highly-efficient CHO cell line generation system with engineered SV40E promoter

Chinese hamster ovary (CHO) cells have been one of the most widely used host cells for the manufacture of therapeutic recombinant proteins. An effective and efficient clinical cell line development process, which could quickly identify those rare, high-producing cell lines among a large population of low and non-productive cells, is of considerable interest to speed up biological drug development. In the glutamine synthetase (GS)-CHO expression system, selection of top-producing cell lines is based on controlling the balance between the expression level of GS and the concentration of its specific inhibitor, l-methionine sulfoximine (MSX). The combined amount of GS expressed from plasmids that have been introduced through transfection and the endogenous CHO GS gene determine the stringency and efficiency of selection. Previous studies have shown significant improvement in selection stringency by using GS-knockout CHO cells, which eliminate background GS expression from the endogenous GS gene in CHOK1SV cells. To further improve selection stringency, a series of weakened SV40E promoters have been generated and used to modulate plasmid-based GS expression with the intent of manipulating GS-CHO selection, finely adjusting the balance between GS expression and GS inhibitor (MSX) levels. The reduction of SV40E promoter activities have been confirmed by TaqMan RT-PCR and GFP expression profiling. Significant productivity improvements in both bulk culture and individual clonal cell line have been achieved with the combined use of GS-knockout CHOK1SV cells and weakened SV40E promoters driving GS expression in the current cell line generation process. The selection stringency was significantly increased, as indicated by the shift towards higher distribution of producing-cell populations, even with no MSX added into cell culture medium. The potential applications of weakened SV40E promoter and GS-knockout cells in development of targeted integration and transient CHO expression systems are also discussed.     

丙肝病毒核心基因靶向性M1GS 核酶的体外抗病毒活性

[目的]丙型肝炎病毒(Hepatitis C Virus,HCV)是引起病毒性肝炎的重要病原.目前临床HCV感染多采用干扰素联合病毒唑进行治疗,但其应答率低且感染易反复,探索新型抗HCV治疗策略与药物具有紧迫的现实意义.[方法]基于大肠埃希菌来源的核糖核酸酶P(RNase P),针对HCV核心基因的序列,设计一小段与之互补的外部引导序列(Guide Sequence,GS),通过PCR将其共价连接至大肠埃希菌RNase P催化性亚基(M1 RNA)的3'末端,从而构建一种靶向性的核酶——M1GS.[结果]构建的M1 GS-HCV/C52核酶在体外可对靶RNA片段产生特异性切割;在HCV感染的Huh7.5.1细胞,该人工核酶也能够显著抑制HCV核心基因的表达,并使培养上清中HCV RNA的拷贝数减少约1500倍.[结论]本研究构建的M1GS-HCV/C52核酶具有显著的体外抗病毒活性,从而为HCV的治疗研究提供了一条潜在途径.