黄花苜蓿铁蛋白cDNA的克隆与序列分析

为克隆黄花苜蓿铁蛋白基因,根据已报道的植物铁蛋白基因序列设计引物,利用RT-PCR法扩增得到大小约为750 bp的特异性片段,并将其克隆到pBlueskript II SK+上.序列分析结果表明,黄花苜蓿铁蛋白基因cDNA全长为756 bp,编码252个氨基酸,与紫花苜蓿铁蛋白基因的核苷酸、氨基酸的同源性均为97%.该基因编码的蛋白质是一个定位在叶绿体上的球形蛋白,理论相对分子质量为28 ku,等电点为5.47,二级结构为α/β混合型蛋白,某些氨基酸在密码子的选择上存在一定的偏向性.该蛋白具有1个FER-RITIN-LIKE功能区、2个铁蛋白基因家族铁结合区域的信号序列、3个潜在的N-糖基化位点、1个蛋白激酶C磷酸化位点、1个依赖于cAMP和cGMP的蛋白激酶磷酸化位点以及3个酪蛋白激酶Ⅱ磷酸化位点等重要的功能基序.黄花苜蓿铁蛋白基因的克隆,为今后利用该基因进行改良植物铁营养成分及提高植物抗氧化胁迫能力的基因工程奠定了基础.     

The Size Flexibility of Ferritin Nanocage Opens a New Way to Prepare Nanomaterials

Ferritins are ubiquitous iron storage proteins where Fe(II) sequestration prevents not only its spontaneous oxidation to Fe(III) but also production of toxic free radicals. Recently, scientists have subverted these nature functions and used ferritin cage structures of nanometer dimensions for encapsulation of guest molecules such as anti‐cancer drugs or bioactive nutrients based on pH induced ferritin disassembly and reassembly property. However, prior to this study, ferritin nanocage was required to disassemble only under harsh pH conditions (≤2.0 or ≥11.0), followed by reassembly at near neutral pH. Such harsh conditions can cause protein or guest molecules damage to a great extent during this pH‐induced unfolding–refolding process. Here, we provide evidence demonstrating that the apoferritin shell is flexible rather than rigid. Indeed, we found that two large complex molecules, uranyl acetate dihydrate and phosphotungstic acid, can reach the cavity of both plant and animal apoferritin followed by mineralization. Moreover, large organic compound such as curcumin and doxorubicin can also be encapsulated within ferritin cavity by its mixing with protein. This strategy will increase the use of ferritin in nanotechnology, and could be also applicable to other shell‐like proteins as templates to prepare nanomaterials.     

Quantum Dots Decorated with Magnetic Bionanoparticles

A new class of water‐soluble, fluorescent, magnetic quantum dots––magnetoferritin bioconjugate––is prepared. The nanostructures are patterned mainly as dimer particles as characterized by high‐angle annular dark‐field scanning transmission electron microscopy and electron energy loss spectroscopy. Magnetic (high spontaneous magnetization values, superparamagnetism) and fluorescent (narrow emission peaks, uniform brightness) properties of both nanoblocks are maintained in the final nanostructure.     

Technical note: measurement of ferritin in bovine milk and its clinical significance

A quantitative ELISA was developed for bovine milk ferritin with an assay limit of 0.16 ng/mL of bovine spleen ferritin. Ferritin-binding activity was detected in bovine milk samples, and this binding activity was inhibited by increasing ionic strength with the addition of 0.5 M (NH₄)₂SO₄. Heat treatment (60°C, 20 min) of bovine milk in the presence of 0.5 M (NH₄)₂SO₄ resulted in a 15 to 58% increase in ferritin concentrations compared with untreated samples. Although the recovery of bovine spleen ferritin added to milk was still low (55 to 90%), even in the presence of increased ionic strength with 0.5 M (NH₄)₂SO₄, recovery was improved by heat treatment at 60°C for 20 min (92 to 95%). Milk ferritin concentrations in 30 milk samples from quarters of 25 cows with mastitis (mean ± SE: 134.2 ± 28.7 ng/mL) were significantly higher than those in 17 quarter milk samples from 17 noninfected lactating cows (7.2 ± 1.2 ng/mL), suggesting that bovine milk contains putative ferritin-binding proteins that inhibit immunoassay for milk ferritin and that bovine milk ferritin is an indicator of IMI.     

较长期喂养转人乳铁蛋白全乳粉对SD 大鼠血清铁、铁蛋白含量的影响

目的：饲喂SD 大鼠转人乳铁蛋白全乳粉90d,监测其对大鼠营养状况以及生理、血液生化指标的影响,从而评价转人乳铁蛋白全乳粉在较长期喂养实验动物时对其血清铁、血清铁蛋白含量的影响。方法：参考中华人民共和国农业行业标准《转基因植物及其产品食用安全检测--大鼠 90 天喂养实验》(NY/T 1102 - 2006)进行。将140 只SD 大鼠按体质量随机分为7 组,每组20 只,雌雄各半,分别为：常规饲料对照组,转基因奶粉低、中、高剂量组和非转基因奶粉低、中、高剂量组。90d 后,观察各组实验动物体质量、食物利用率以及血生化指标。结果: 转人乳铁蛋白全乳粉经过90d 喂养实验动物后,各组动物生长发育良好,含转人乳铁蛋白全乳粉与不含转人乳铁蛋白全乳粉组相比,大鼠的体质量与食物利用率、总进食量均无显著差异(P ≥ 0.05),在较长期喂养过程中未观察到含转人乳铁蛋白全乳粉对大鼠产生不良作用,含转人乳铁蛋白全乳粉可显著增加血清铁和铁蛋白水平(P ≤0.05)。结论：SD 大鼠较长期食用转人乳铁蛋白全乳粉未出现安全问题,且可以提高实验动物机体铁的营养状况。     

In vitro iron absorption of α-lactalbumin hydrolysate-iron and β-lactoglobulin hydrolysate-iron complexes

To study the feasibility of promoting iron absorption by peptides derived from α-lactalbumin and β-lactoglobulin, the present work examined the transport of iron across Caco-2 monolayer cell as in vitro model. Caco-2 cells were seeded in bicameral chambers with α-lactalbumin hydrolysate-Fe (α-LAH-Fe) complex and β-lactoglobulin hydrolysate-Fe (β-LGH-Fe) complex, α-LAH and iron mixture, β-LGH and iron mixture, FeSO₄ and ascorbic acid mixture, and FeSO₄. In addition, the cytotoxicity of α-LAH-Fe and β-LGH-Fe complexes were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The iron absorption and ferritin content were assessed using the coupled in vitro digestion/Caco-2 cell model. Results support that peptide-iron complexes can promote ferritin formation and it is possible to apply β-LGH-Fe complexes as iron-fortified supplements with high iron absorbability.     

Hierarchically Ordered Supramolecular Protein-Polymer Composites with Thermoresponsive Properties

Synthetic macromolecules that can bind and co-assemble with proteins are important for the future development of biohybrid materials. Active systems are further required to create materials that can respond and change their behavior in response to external stimuli. Here we report that stimuli-responsive linear-branched diblock copolymers consisting of a cationic multivalent dendron with a linear thermoresponsive polymer tail at the focal point, can bind and complex Pyrococcus furiosus ferritin protein cages into crystalline arrays. The multivalent dendron structure utilizes cationic spermine units to bind electrostatically on the surface of the negatively charged ferritin cage and the in situ polymerized poly(di(ethylene glycol) methyl ether methacrylate) linear block enables control with temperature. Cloud point of the final product was determined with dynamic light scattering (DLS), and it was shown to be approximately 31 °C at a concentration of 150 mg/L. Complexation of the polymer binder and apoferritin was studied with DLS, small-angle X-ray scattering, and transmission electron microscopy, which showed the presence of crystalline arrays of ferritin cages with a face-centered cubic (fcc Fm3¯m) Bravais lattice where lattice parameter a = 18.6 nm. The complexation process was not temperature dependent but the final complexes had thermoresponsive characteristics with negative thermal expansion.     

Carotenoids,but Not Vitamin A,Improve Iron Uptake and Ferritin Synthesis by Caco‐2 Cells from Ferrous Fumarate and NaFe‐EDTA

Due to the high prevalence of iron and vitamin A deficiencies and to the controversy about the role of vitamin A and carotenoids in iron absorption, the objectives of this study were to evaluate the following: (1) the effect of a molar excess of vitamin A as well as the role of tannic acid on iron uptake by Caco‐2 cells; (2) iron uptake and ferritin synthesis in presence of carotenoids without pro‐vitamin A activity: lycopene, lutein, and zeaxantin; and (3) iron uptake and ferritin synthesis from ferrous fumarate and NaFe‐EDTA. Cells were incubated 1 h at 37 °C in PBS pH 5.5, containing ⁵⁹Fe and different iron compounds. Vitamin A, ferrous fumarate, β‐carotene, lycopene, lutein, zeaxantin, and tannic acid were added to evaluate uptake. Ferritin synthesis was measured 24 h after uptake experiments. Vitamin A had no effect on iron uptake by Caco‐2 cells, and was significantly lower from NaFe‐EDTA than from ferrous fumarate (15.2 ± 2.5 compared with 52.5 ± 8.3 pmol Fe/mg cell protein, respectively). Carotenoids increase uptake up to 50% from fumarate and up to 300% from NaFe‐EDTA, since absorption from this compound is low when administered alone. We conclude the following: (1) There was no effect of vitamin A on iron uptake and ferritin synthesis by Caco‐2cells. (2) Carotenoids significantly increased iron uptake from ferrous fumarate and NaFe‐EDTA, and were capable of partially overcoming the inhibition produced by tannic acid. (3) Iron uptake by Caco‐2 cell from NaFe‐EDTA was significantly lower compared to other iron compounds, although carotenoids increased and tannic acid inhibited iron uptake comparably to ferrous fumarate.