植物蛋白质S-亚硝基化修饰的检测与分析

S-亚硝基化是一种重要的蛋白质翻译后修饰方式, 是指一氧化氮(NO)基团共价连接至靶蛋白特定半胱氨酸残基的自由巯基, 从而形成S-亚硝基硫醇(SNO)的过程。S-亚硝基化修饰广泛存在于各有机体中, 通过改变蛋白质生化活性、稳定性、亚细胞定位以及蛋白质-蛋白质相互作用等机制而调控不同的生物学过程或信号通路。在蛋白质S-亚硝基化检测分析方法中, 最为广泛使用的是生物素转化法(biotin switch assay), 其基本原理是首先封闭未被修饰的自由巯基, 进而将被修饰的SNO基团特异地还原为自由巯基并使用生物素将其特异标记。被生物素标记的半胱氨酸残基(即被修饰位点)可进一步通过蛋白质免疫印迹和/或质谱等方法进行检测分析。该文详细描述了植物蛋白质样品的体内和体外生物素转化法的实验流程, 并对实验过程中的注意事项进行了讨论。     

双拷贝人α型心钠素基因的组建及大肠杆菌分泌型克隆与表达

将单拷贝人α心钠素基因3′端用Ban Ⅱ酶解除去包括终止密码在内的36个碱基对,代之以人工合成的含Glu-Lys-Phe-Glu连接片段与另一单拷贝人α心钠素基因的5′端串连成编码60肽的双拷贝心钠素基因,克隆于大肠杆菌分泌型表达载体pIN-Ⅲ-OmpA_2质粒中,表达生成60肽的双拷贝人α型心钠素衍生物,在信号肽的作用下分泌至胞膜间质并自动切割为60肽的外源基因产物。分子量约8K的表达产物用分子筛或超滤膜分离后再经HPLC纯化,表达产物具有明显的心钠素放免活性和舒张血管活性。     

Kininogen and Kinin in Experimental Spinal Cord Injury

Activation of the kallikrein-kinin system has been implicated in the pathogenesis of vasogenic brain edema and posttraumatic vascular injury. We determined the levels of kininogen and kinin in an experimental spinal cord injury model in the rat. Kininogen content in traumatized cord segments increased in a time-dependent manner. Western blot analysis showed that the kininogen in traumatized cord comigrates with 68K low-molecular-weight kininogen or T-kininogen. Trypsin treatment of the kininogen in traumatized cord released both bradykinin and T-kinin, which were separated by HPLC and quantified with a kinin radioimmunoassay. Endogenous kinin levels in the frozen spinal cord also increased up to 40-fold 2 h after injury as compared with controls. The results demonstrate an increased accumulation of kininogen and its conversion to vasoactive kinins in experimental spinal cord injury.     

湖北省泽泻科、水鳖科、眼子菜科及茨藻科植物花粉形态研究

本文利用光镜及扫描电镜对湖北省泽泻科、水鳖科、眼子菜科及茨藻科11属29种1变种1变型植物(另加采于湛江的软骨草)的花粉形态进行了研究,发现泽泻科植物花粉具多个圆形萌发孔,外壁表面为小刺状纹饰;茨藻科植物花粉具远极单槽,表面为绉波状纹饰;眼子菜科及本文研究的水鳖科植物花粉均无萌发孔,分别具网状和小刺状饰纹饰。1.茨藻科植物花粉最原始,泽泻科花粉较进化,眼子菜科花粉较水鳖科花粉进化;2.泽泻属与泽苔草属花粉较慈姑属花粉原始;3.鞘叶眼子菜亚屈花粉较眼子菜亚属的花粉处于更高演化阶段;4.多孔茨藻花粉在该科中最原始。本文工作尚对易变形水生植物花粉形态研究方法进行了尝试。     

氧合血红蛋白Fe-O2键合态 ab initio 研究

本文报道用自旋非限制Hartree-Fock方法(spin unrestricted Hartree-Fock method简写UHF)对氧合血红蛋白的Fe-O₂键合态进行ab initio研究。结果表明Fe^(Ⅱ)、Oc和Or的Mulliken布居值分别为24.18、8.19和7.64。这说明氧合血红蛋白的Fe-O₂键合态不发生电子转移。研究模型所得到的频率与实验频率基本一致。研究结果不支持Weiss提出的Fe³⁺O₂^-模型,为解释血红蛋白传输氧的作用机理提供了新的理论依据。     

江苏菜豆同工凝集素的分离纯化及性质研究

江苏菜豆经酸水(PH2.0)抽提,硫酸铵分级沉淀,分离植物血球凝集素(PHA-P),分子量为128000的糖蛋白,活性回收率在80％以上,PHA-P经SP-sephadexc-50离子交换层析,分成L_4,L_3E_1,L_2E_2,L_1E_3,和E_4同工凝集素。 L_4和E_4等电点为5.4和6.5。亚基分子量分别是31000和33000,并有类似的氨基酸组成。PAGE分析为单一蛋白带。红细胞凝集活性随电泳迁移速度的加快而增强,促淋性细胞分裂活性则减弱。E_4血凝活性受CalNAc,EDTA抑制和Zn~(++)的促进。     

Circular RNA AFF4 modulates osteogenic differentiation in BM-MSCs by activating SMAD1/5 pathway through miR-135a-5p/FNDC5/Irisin axis

Bone marrow-derived mesenchymal stem cells (BM-MSCs), the common progenitor cells of adipocytes and osteoblasts, have been recognized as the key mediator during bone formation. Herein, our study aim to investigate molecular mechanisms underlying circular RNA (circRNA) AFF4 (circ_AFF4)-regulated BM-MSCs osteogenesis. BM-MSCs were characterized by FACS, ARS, and ALP staining. Expression patterns of circ_AFF4, miR-135a-5p, FNDC5/Irisin, SMAD1/5, and osteogenesis markers, including ALP, BMP4, RUNX2, Spp1, and Colla1 were detected by qRT-PCR, western blot, or immunofluorescence staining, respectively. Interactions between circ_AFF4 and miR-135a-5p, FNDC5, and miR-135a-5p were analyzed using web tools including TargetScan, miRanda, and miRDB, and further confirmed by luciferase reporter assay and RNA pull-down. Complex formation between Irisin and Integrin αV was verified by Co-immunoprecipitation. To further verify the functional role of circ_AFF4 in vivo during bone formation, we conducted animal experiments harboring circ_AFF4 knockdown, and born samples were evaluated by immunohistochemistry, hematoxylin and eosin, and Masson staining. Circ_AFF4 was upregulated upon osteogenic differentiation induction in BM-MSCs, and miR-135a-5p expression declined as differentiation proceeds. Circ_AFF4 knockdown significantly inhibited osteogenesis potential in BM-MSCs. Circ_AFF4 stimulated FNDC5/Irisin expression through complementary binding to its downstream target molecule miR-135a-5p. Irisin formed an intermolecular complex with Integrin αV and activated the SMAD1/5 pathway during osteogenic differentiation. Our work revealed that circ_AFF4, acting as a sponge of miR-135a-5p, triggers the promotion of FNDC5/Irisin via activating the SMAD1/5 pathway to induce osteogenic differentiation in BM-MSCs. These findings gained a deeper insight into the circRNA-miRNA regulatory system in the bone marrow microenvironment and may improve our understanding of bone formation-related diseases at physiological and pathological levels.Subject terms: Stem cells, Diseases     

Different Glycosylation in Acetylcholinesterases from Mammalian Brain and Erythrocytes

Acetylcholinesterases (EC 3.1.1.7, AChE) have varying amounts of carbohydrates attached to the core protein. Sequence analysis of the known primary structures gives evidence for several asparagine-linked carbohydrates. From the differences in molecular mass determined on sodium dodecyl sulfate-polyacrylamide gel before and after deglycosylation with N-glycosidase F (EC 3.2.2.18), it is seen that dimeric AChE from red cell membranes is more heavily glycosylated than the tetrameric brain enzyme. Furthermore, dimeric and tetrameric forms of bovine AChE are more heavily glycosylated than the corresponding human enzymes. Monoclonal antibodies 2E6, 1H11, and 2G8 raised against detergent-soluble AChE from electric organs of Torpedo nacline timilei as well as Elec-39 raised against AChE from Electrophorus electricus cross-reacted with AChE from bovine and human brain but not with AChE from erythrocytes. Treatment of the enzyme with N-glycosidase F abolished binding of monoclonal antibodies, suggesting that the epitope, or part of it, consists of N-linked carbohydrates. Analysis of N-acetylglucosamine sugars revealed the presence of N-acetylglucosamine in all forms of cholinesterases investigated, giving evidence for N-linked glycosylation. On the other hand, N-acetylgalactosamine was not found in AChE from human and bovine brain or in butyrylcholinesterase (EC 3.1.1.8) from human serum, indicating that these forms of cholinesterase did not contain O-linked carbohydrates. Despite the notion that within one species, the different forms of AChE arise from one gene by different splicing, our present results show that dimeric erythrocyte and tetrameric brain AChE must undergo different postsynthetic modifications leading to differences in their glycosylation patterns.