NEDD4L binds the proteasome and promotes autophagy and bortezomib sensitivity in multiple myeloma

Multiple myeloma (MM) remains an incurable plasma cell cancer characterized by abnormal secretion of monoclonal immunoglobulins. The molecular mechanism that regulates the drug sensitivity of MM cells is being intensively studied. Here, we report an unexpected finding that the protein encoded by neural precursor cell-expressed developmentally downregulated gene 4L (NEDD4L), which is a HECT E3 ligase, binds the 19S proteasome, limiting its proteolytic function and enhancing autophagy. Suppression of NEDD4L expression reduced bortezomib (Bor) sensitivity in vitro and in vivo, mainly through autophagy inhibition mediated by low NEDD4L expression, which was rescued by an autophagy activator. Clinically, elevated expression of NEDD4L is associated with a considerably increased probability of responding to Bor, a prolonged response duration, and improved overall prognosis, supporting both the use of NEDD4L as a biomarker to identify patients most likely to benefit from Bor and the regulation of NEDD4L as a new approach in myeloma therapy.Subject terms: Myeloma, Protein quality control     

Exosomes in atherosclerosis: Convergence on macrophages

As the primary cells of atherosclerotic plaques, macrophages play a central role in the occurrence and progression of atherosclerosis (AS). In recent years, macrophages have received extensive attention as therapeutic targets. Exosomes, as natural nanoparticles, have high biocompatibility and strong targeting ability and have been widely studied as imaging agents and drug carriers. Studies on the relationship between atherosclerotic macrophages and exosomes have been focused on for the past few years. Nevertheless, no complex review has been undertaken in this area. In this review, we summarize in detail the role of macrophages in atherosclerosis, especially their plasticity and phenotypic and distributional heterogeneity. Based on the high correlation between macrophages and the pathological process of atherosclerosis, as well as the targeting of exosomes, we further review the clinical application of targeting macrophage-associated exosomes. We focus on the role of macrophage-associated exosomes in the phenotypic transformation of cells in atherosclerosis, providing a new idea for the clinical application of targeting macrophage-associated exosomes. Finally, we specifically summarize and prospect the diagnosis of macrophage-associated exosomes, such as imaging agent delivery, biomarkers and therapeutic strategies.     

木质素芳香族化合物降解菌Sphingobium sp. SYK-6的研究进展

木质素是木质的主要成分之一,在自然界中,高分子木质素被真菌的胞外酶分解成低分子芳香族化合物,然后土壤细菌将其完全降解为二氧化碳。由此可见,木质素的完全降解过程是真菌和细菌的共同作用。研究细菌的降解机制,一方面可以理解芳香族化合物在生态系中的碳素循环,另一方面可以为木质素的有效利用提供基因和酶工具,将可再生资源的木质素转化成高附加价值的工业产品。Sphingobium sp.SYK-6是1987年从造纸厂废水中以木质素中的联苯化合物(5,5’-脱氢联香草酸)作为唯一碳源分离出的木质素化合物降解菌。在长达25年以上的研究中我们阐明了一系列芳香族化合物的代谢途径,克隆了相关基因,2012年随着基因组测序的完成,整个降解功能的全貌展现出来。介绍内容:(1)基因组信息;(2)芳醚化合物代谢;(3)联苯化合物代谢;(4)阿魏酸代谢;(5)木质素化合物降解过程中四氢叶酸依赖型机制;(6)原儿茶酸4,5开环途径;(7)3-甲氧基没食子酸代谢的多样性;(8)应用研究。我们希望SYK-6菌株成为一个让人们理解木质素化合物降解的模式菌株。最后结合课题组现在的研究课题展望了木质素化合物的降解研究的发展方向。     

Ⅱ型志贺毒素单克隆抗体可变区基因克隆及单链抗体的表达和功能鉴定

目的从分泌抗肠出血性大肠埃希菌Ⅱ型志贺毒素中和单克隆抗体杂交瘤细胞株S2C4中克隆抗体可变区基因,构建单链抗体（ScFv）,进行原核表达,并对其功能进行鉴定。方法从杂交瘤细胞株S2C4中提取总RNA,逆转录成cDNA。在cDNA3’-OH末端添加poly．G。PCR扩增包括5’非翻译区和信号肽序列在内的抗体重、轻链可变区基因VH和VL,PCR产物装入T—A载体测序。根据测序结果,设计引物分别扩增VH和VL编码区,再通过重叠PCR,在VH和VL．编码区基因之间引入连接链,构建Scn基因,并克隆到表达载体pComb3xSS中。重组载体导入E．coliTop10F’进行表达,重组蛋白经纯化后,分别用ELISA和动物保护性实验鉴定其生物学活性。结果VH和VL编码区基因全长分别为396bp和378bp,ScFv基因能在大肠埃希菌中高效表达,表达产物的分子量为34000,用NiSO4亲和层析柱成功纯化。功能性实验表明纯化的重组蛋白可以与Stx2毒素有效结合,能保护动物抵御毒素分子的攻击。结论成功地克隆S2C4单抗可变区基因,并构建、表达其单链抗体ScFv,为下一步进行该抗体人源化奠定实验基础。     

树干径流模型

以红松代表针叶树,栎树代表阔叶树,通过模拟实验研究树干径流过程,根据实验结果从机理上构造了模型。树干径流模型为一个方程组用“辗转迭代法”求出了数值解。该方程组基本上揭示了树干径流的规律,从实验与模拟结果对比分析中得到了佐证。     

秦岭火地塘林区油松林土壤碳循环研究

采用土壤碳循环分室模型,对秦岭火地塘林区油松林土壤碳各分室的碳贮量和通量进行了研究.结果表明,研究区油松林土壤有机碳贮量为146.071 t·hm^-2,其中矿质土壤层130.366 t·hm^-2、凋落物层12.626 t·hm^-2,土壤有机碳储存量低于我国森林土壤碳贮量平均值,高于处在我国最低水平的暖性针叶林和热带林,与本区锐齿栎林相比也明显偏低.林地植物年凋落进入土壤的碳量为5939 t·hm^-2,其中地上枯枝落叶占56.9%、地下死细根占43.1%; 凋落物层分解后每年以腐殖酸形式输入矿质土壤中的碳量为2.034 t·hm^-2.油松林土壤(含植物根系)年呼吸释放碳量14.012 t·hm^-2,其中凋落物层、矿质土壤层、死根系和活根系分别占林地总呼吸量的15.7%、14.5%、11.7%和58.1%.     

A tightly packed hydrophobic cluster directs the formation of an off-pathway sub-millisecond folding intermediate in the alpha subunit of tryptophan synthase, a TIM barrel protein

Protein misfolding is now recognized as playing a crucial role in both normal and pathogenic folding reactions. An interesting example of misfolding at the earliest state of a natural folding reaction is provided by the alpha-subunit of tryptophan synthase, a (beta/alpha)(8) TIM barrel protein. The molecular basis for the formation of this off-pathway misfolded intermediate, I(BP), and a subsequent on-pathway intermediate, I1, was probed by mutational analysis of 20 branched aliphatic side-chains distributed throughout the sequence. The elimination of I(BP) and the substantial destabilization of I1 by replacement of a selective set of the isoleucine, leucine or valine residues (ILV) with alanine in a large ILV cluster external-to-the-barrel and spanning the N and C termini (cluster 2) implies tight-packing at most sites in both intermediates. Differential effects on I(BP) and I1 for replacements in alpha3, beta4 and alpha8 at the boundaries of cluster 2 suggest that their incorporation into I1 but not I(BP) reflects non-native folds at the edges of the crucial (beta/alpha)(1-2)beta(3) core in I(BP). The retention of I(BP) and the smaller and consistent destabilization of both I(BP) and I1 by similar replacements in an internal-to-the-barrel ILV cluster (cluster 1) and a second external-to-the-barrel ILV cluster (cluster 3) imply molten globule-like packing. The tight packing inferred, in part, for I(BP) or for all of I1 in cluster 2, but not in clusters 1 and 3, may reflect the larger size of cluster 2 and/or the enhanced number of isoleucine, leucine and valine self-contacts in and between contiguous elements of secondary structure. Tightly packed ILV-dominated hydrophobic clusters could serve as an important driving force for the earliest events in the folding and misfolding of the TIM barrel and other members of the (beta/alpha)(n) class of proteins.     

利用RNA干扰抑制HeLa细胞中CTCF基因的表达

目的:CTCF是一种多功能的转录因子,参与启动子调控、绝缘子功能和遗传印记等过程。利用RNA干扰技术抑制CTCF在体细胞中的表达。方法:以CTCF为靶基因,利用ShortCutRNaseⅢ切割体外转录的长双链RNA制备esiRNA,转染HeLa细胞,用RTReal-TimePCR检测CTCF的mRNA水平,用免疫印迹检测CTCF水平。结果:转染CTCFesiRNA后,细胞内的CTCFmRNA被抑制了70%左右,CTCF水平明显下降。结论:CTCF的esiRNA可以明显抑制CTCF在HeLa细胞中的表达,从而为进一步研究CTCF的未知功能打下了基础。     

耐碱性木聚糖酶产酶菌株的选育

目的:筛选出产碱性木聚糖酶酶活力高的菌株。方法:从碱性环境中采集样品,以自制木聚糖为惟一利碳源,采用刚果红透明圈法于摇瓶发酵法相结合筛选出18株产木聚糖酶酶活较高的菌株,其中1株酶活最高可达335.14 IU/ml。结果:通过培养特性及形态特征初步鉴定为芽孢杆菌。对部分酶学性质研究的结果表明:其作用最适温度60℃,最适pH8.0,在pH9.0条件下仍具有80%酶活力。结论:属于耐碱性木聚糖酶,具有很好的工业应用前景。