Phospholipid,Nature's own slide and cover slip for cryo-electron microscopy

Thin films of surface-active compounds, with or without particulate material, can be obtained by immersing and withdrawing a bare specimen grid from a solution/suspension of the compound. Immediately after withdrawing the grid, thinning of the film starts. Thinning is initially powered by gravity and capillary forces and will proceed in thin films (< 100 nm) driven by intermolecular forces until the London-van der Waals attractive forces come to an equilibrium with electrostatic repulsion of similarly charged surfaces of the film. With small unilamellar vesicles prepared from the phospholipid dimyristoyl phosphatidyl choline (DMPC) the draining behaviour of these films was studied by cryo-electron microscopy. Small unilamellar vesicles were observed within the film as well as the coalescence of these vesicles into sheets (‘leaky’ membrane fusion). Sheets dominate the images when films are allowed to drain for longer periods (>3min). Thin films were formed on grids from catalase crystals suspended in a DMPC suspension and vitrified by cooling. High-resolution information was obtained by electron diffraction at low temperature and under low-dose conditions from catalase crystals surrounded by small vesicles as well as from catalase crystals surrounded by sheets of DMPC. In the latter case the water content drops from 99% (DMPC in small vesicles) to less than 30% (DMPC in sheets) during draining. Ferritin was added to a DMPC suspension and thin films were prepared and vitrified. After prolonged draining ferritin molecules were deposited in layers with a stepwise increase in thickness. Draining of thin films has thus a dehydrating effect as well as a sorting and ordering effect. These effects must be considered when using surface-active compounds at air-water interfaces as a slide and cover slip for electron microscopy.     

LC3/FtMt Colocalization Patterns Reveal the Progression of FtMt Accumulation in Nigral Neurons of Patients with Progressive Supranuclear Palsy

Mitochondrial ferritin (FtMt) is a mitochondrial iron storage protein associated with neurodegenerative diseases. In patients with progressive supranuclear palsy (PSP), FtMt was shown to accumulate in nigral neurons. Here, we investigated FtMt and LC3 in the post-mortem midbrain of PSP patients to reveal novel aspects of the pathology. Immunohistochemistry was used to assess the distribution and abnormal changes in FtMt and LC3 immunoreactivities. Colocalization analysis using double immunofluorescence was performed, and subcellular patterns were examined using 3D imaging and modeling. In the substantia nigra pars compacta (SNc), strong FtMt-IR and LC3-IR were observed in the neurons of PSP patients. In other midbrain regions, such as the superior colliculus, the FtMt-IR and LC3-IR remained unchanged. In the SNc, nigral neurons were categorized into four patterns based on subcellular LC3/FtMt immunofluorescence intensities, degree of colocalization, and subcellular overlapping. This categorization suggested that concomitant accumulation of LC3/FtMt is related to mitophagy processes. Using the LC3-IR to stage neuronal damage, we retraced LC3/FtMt patterns and revealed the progression of FtMt accumulation in nigral neurons. Informed by these findings, we proposed a hypothesis to explain the function of FtMt during PSP progression.     

H-Ferritin Produced by Myeloid Cells Is Released to the Circulation and Plays a Major Role in Liver Iron Distribution during Infection

During infections, the host redistributes iron in order to starve pathogens from this nutrient. Several proteins are involved in iron absorption, transport, and storage. Ferritin is the most important iron storage protein. It is composed of variable proportions of two peptides, the L- and H-ferritins (FTL and FTH). We previously showed that macrophages increase their expression of FTH1 when they are infected in vitro with Mycobacterium avium, without a significant increase in FTL. In this work, we investigated the role of macrophage FTH1 in M. avium infection in vivo. We found that mice deficient in FTH1 in myeloid cells are more resistant to M. avium infection, presenting lower bacterial loads and lower levels of proinflammatory cytokines than wild-type littermates, due to the lower levels of available iron in the tissues. Importantly, we also found that FTH1 produced by myeloid cells in response to infection may be found in circulation and that it plays a key role in iron redistribution. Specifically, in the absence of FTH1 in myeloid cells, increased expression of ferroportin is observed in liver granulomas and increased iron accumulation occurs in hepatocytes. These results highlight the importance of FTH1 expression in myeloid cells for iron redistribution during infection.     

铁蛋白铁释放机理的研究进展

铁蛋白是一种广泛存在于生命体中的铁贮藏蛋白,具有调节机体铁代谢平衡和去除二价铁毒性的双重功能。缺铁严重影响着全球近一半人的健康,研究表明,铁蛋白具有良好的补铁活性而且安全、高效,能够取代具有毒副作用的传统补铁试剂。因此,寻求并开发以铁蛋白为原料的新型补铁功能食品已成为一种趋势。为了更科学地应用于实践和开发,对铁蛋白理化性质及其生物学功能的阐明显得颇为重要。目前,关于铁蛋白铁释放机理的研究分为体外和体内两个方面,体外机理涉及还原剂和螯合剂的共同作用,而体内机理主要涉及降解途径和酶介导的还原释放途径。综述了国内外有关铁蛋白铁释放机理的研究进展,以期为新型补铁功能食品的开发提供理论依据。     

基因工程纳米颗粒在EB病毒疫苗研发中的应用概述

EB(Epstein-Barr)病毒普遍性感染人类并引发多种病毒相关性癌症,其中鼻咽癌与EB病毒感染相关性最高,并在我国广东等地区发病率极高,是我国重点防治的十大恶性肿瘤之一.截至目前,没有任何一款上市可接种的EB病毒防治性疫苗,针对EB病毒相关疾病的免疫治疗手段在临床试验中也收效甚微.近年来,纳米颗粒基因改造技术的飞...     

生物模板法制备纳米Ni粒子的研究

研究了沉积环境、pH值、沉积时间、铁蛋白浓度对生物模板法制备纳米Ni粒子的影响。研究结果表明, 最佳制备条件为: 在N₂保护环境下, 向含2 mg/mL脱铁铁蛋白, pH=8.0的Tris缓冲溶液中加入Ni(Ac)₂, 沉积2.5 h后, 加入还原剂NaBH₄, 得到黑色溶液。将溶液在pH=8.0的Tris缓冲溶液中透析除杂, 就可以制备出黑灰色的含Ni的铁蛋白溶液。利用Zeta激光粒度仪测试、TEM检测和EDX分析, 表明制备的纳米Ni样品具有平均粒径5 nm, 粒度分布均匀, 分散性好的特点。     

橙皮素与铁蛋白的共价相互作用及其对铁蛋白理化性质的影响

以缺铁的铁蛋白和橙皮素为原料,在pH 9条件下制备铁蛋白-橙皮素共价复合物,探讨共价结合对蛋白结构和理化性质的影响。结果表明,橙皮素与铁蛋白的共价作用降低了铁蛋白的荧光强度并产生红移现象,改变了铁蛋白的结构。铁蛋白-橙皮素共价复合物的溶解度改变,等电点降低,其铁氧化沉淀活性提高,铁的还原释放活性降低。同时,共价复合物的热稳定性增强。铁蛋白-橙皮素复合物的形成赋予了铁蛋白新的功能特性,对探究食品组分相互作用,开发基于铁蛋白的相关食品或药品具有一定的理论指导。     

Genetically Modified Ferritin Nanoparticles with Bone-Targeting Peptides for Bone Imaging

Bone homeostasis plays a major role in supporting and protecting various organs as well as a body structure by maintaining the balance of activities of the osteoblasts and osteoclasts. Unbalanced differentiation and functions of these cells result in various skeletal diseases, such as osteoporosis, osteopetrosis, and Paget’s disease. Although various synthetic nanomaterials have been developed for bone imaging and therapy through the chemical conjugation, they are associated with serious drawbacks, including heterogeneity and random orientation, in turn resulting in low efficiency. Here, we report the synthesis of bone-targeting ferritin nanoparticles for bone imaging. Ferritin, which is a globular protein composed of 24 subunits, was employed as a carrier molecule. Bone-targeting peptides that have been reported to specifically bind to osteoblast and hydroxyapatite were genetically fused to the N-terminus of the heavy subunit of human ferritin in such a way that the peptides faced outwards. Ferritin nanoparticles with fused bone-targeting peptides were also conjugated with fluorescent dyes to assess their binding ability using osteoblast imaging and a hydroxyapatite binding assay; the results showed their specific binding with osteoblasts and hydroxyapatite. Using in vivo analysis, a specific fluorescent signal from the lower limb was observed, demonstrating a highly selective affinity of the modified nanoparticles for the bone tissue. These promising results indicate a specific binding ability of the nanoscale targeting system to the bone tissue, which might potentially be used for bone disease therapy in future clinical applications.     

热处理对鲟鱼肝脏铁蛋白稳定性的影响

以西伯利亚鲟鱼肝脏为原料，分离纯化出鲟鱼肝脏铁蛋白（liver ferritin of Acipenser baerii，ABLF）。采用电泳、紫外、动态光散射（dynamic light scattering，DLS）、傅里叶变换红外光谱等分析5 个不同热处理温度（60～100 ℃）对ABLF稳定性的影响，同时探究25～65 ℃热处理温度对ABLF铁释放速率快慢的影响及在4、25 ℃条件下ABLF的贮藏稳定性。研究表明：随着热处理温度的升高（60～100 ℃），ABLF的溶解度和铁含量逐渐降低，蛋白聚集体含量增加；红外光谱分析显示ABLF中α-螺旋相对含量逐渐降低，无规卷曲相对含量整体呈增加趋势，以上现象在80～100 ℃范围内尤为明显，而在60～80 ℃范围内变化较小，表明ABLF在60～80 ℃具有良好的热稳定性。ABLF的铁释放速率随温度升高（25～65 ℃）而增加。4 ℃条件下ABLF贮藏稳定性高于25 ℃。本研究将为ABLF的加工及利用提供理论依据。