Efficiency of the monofunctionalized C60 fullerenes as membrane targeting agents studied by all-atom molecular dynamics simulations

Abstract

Transmembrane translocation of C₆₀ fullerenes functionalized by the single amino-derivative in neutral and charged forms was studies by extensive all-atom molecular dynamics simulations. It is shown that these complexes exhibit very strong affinity to the membrane core, but their spontaneous translocation through the membrane is not possible at practical time scale. In contrast, free amino derivatives translocate through the membrane much easier than their complexes with fullerenes, but do not have pronounced affinity to the membrane interior. Our results suggest that monofunctionalized C₆₀ could be extremely efficient membrane targeting agents, which facilitate accumulation of the water-soluble compounds in the hydrophobic core of lipid bilayer.     

Sorting things out through endoplasmic reticulum quality control

Abstract

The endoplasmic reticulum (ER) is a highly organized and specialized organelle optimized for the production of proteins. It is comprised of a highly interconnected network of tubules that contain a large set of resident proteins dedicated to the maturation and processing of proteins that traverse the eukaryotic secretory pathway. As protein maturation is an imperfect process, frequently resulting in misfolding and/or the formation of aggregates, proteins are subjected to a series of evaluation processes within the ER. Proteins deemed native are sorted for anterograde trafficking, while immature or non-native proteins are initially retained in the ER in an attempt to rescue the aberrant products. Terminally misfolded substrates are eventually targeted for turnover through the ER-associated degradation or ERAD pathway to protect the cell from the release of a defective product. A clearer picture of the identity of the machinery involved in these quality control evaluation processes and their mechanisms of actions has emerged over the past decade.     

A site-directed cross-linking approach to the characterization of subunit E-subunit G contacts in the vacuolar H+-ATPase stator

Abstract

To operate as a rotary motor, the ATP-hydrolyzing domain of the vacuolar H⁺-ATPase must be connected to a fixed structure in its membrane-bound proton pump domain by a mechanical stator. Although low-resolution structural data and spectroscopic analysis indicate that a filament-like subunit E/subunit G heterodimer performs this role, more detailed information about the relative arrangement of these subunits is limited. We have used a site-directed cross-linking approach to show that, in both bacterial and yeast V-type ATPases, the N-terminal α-helical segments of the G and E subunits are closely aligned over a distance of up to 40 Å. Furthermore, cross-linking coupled to mass spectrometry shows that the C-terminal end of G is anchored at the C-terminal globular domain of subunit E. These data are consistent with a stator model comprising two ～ 150 Å long parallel α-helices linked to each other at both ends, stabilized by a coiled-coil arrangement and capped by the globular C-terminal domain of E that connects the cytoplasmic end of the helical structure to the V-ATPase catalytic domain.     

Polyethylenimine of various molecular weights as adjuvant for transfection mediated by cationic liposomes

Over the last years significant progress has been made in non-viral gene delivery mediated by cationic liposomes. However, the results obtained are still far from being satisfactory regarding transfection efficiency, particularly when compared to that achieved using viral vectors. We have previously demonstrated that association of transferrin with cationic liposomes significantly improves transfection in a large variety of cells, both in vitro and in vivo. In this work, several strategies have been explored in order to further improve transfection mediated by transferrin-associated lipoplexes. To this regard, the effect on transfection of pre-condensation of DNA with polyethylenimine of low MWs (2.7, 2.0 and 0.8 KDa) at various N/P ratios, lipid composition, cationic lipid/DNA (+/-) charge ratio and the presence of a surfactant in the lipoplexes was investigated. Two different modes for preparing the liposomes were tested and the extent of cell association of their complexes with DNA as well as their capacity to protect the carried DNA were evaluated. Our results show that complexes generated from cationic liposomes prepared by the ethanol injection method in which the carried DNA was pre-condensed with low MW polyethylenimine are highly efficient in mediating transfection. The differential modulating effect observed upon association of transferrin to various liposome formulations on transfection mediated by the polyethylenimine-complexes suggests that these complexes enter into the cells through different pathways (involving clathrin versus caveolin), most likely by taking advantage of their intrinsic biophysical properties to escape from the endosome to the cytosol.     

Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study

The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.     

A molecular systematic investigation of the Acrochaetiales (Florideophycidae,Rhodophyta) and related taxa based on nuclear small-subunit ribosomal DNA sequence data

Small-subunit ribosomal DNA (SSU) sequences for 14 members of the rhodophyte order Acrochaetiales were used to generate phylogenetic trees to determine whether the Acrochaetiales is monophyletic and to assess the relationships of acrochaetioid algae to closely related taxa. Within the Acrochaetiales, two strongly supported groups are recognized: one group that includes, in addition to three other species, the type species of the genera Acrochaetium, Audouinella and Rhodochorton; and a second group that includes an unequivocal representative of Colaconema and nine additional species. The relationships of these groups with the Batrachospermales, Palmariales and Nemaliales were not resolved and the possibility that the Acrochaetiales is polyphyletic was not eliminated. Resolution of relationships for species within these groups was strong in only a few cases, with many of the phylogenetic issues at this level remaining equivocal. Our results do not position Camontagnea in the Acrochaetiales, as has recently been suggested, but strongly ally this genus to the family Rhodothamniellaceae of the Palmariales. The phylogenetic position of Rhododraparnaldia, an alga intermediate between the Acrochaetiales and Batrachospermales, remains unresolved. These molecular results provide a foundation for assessing the taxonomic significance of phenotypic characters (e.g. anatomy, life histories, morphology, phycoerythrin type and plastid details) variously considered taxonomically significant in the Acrochaetiales.     

分子马达生物传感器检测食源性轮状病毒

通过分子马达生物传感器技术建立一种特异、便捷、快速的食源性轮状病毒检测方法.以F0F1-ATPase为核心构建分子马达,以轮状病毒保守片段VP7设计各血清型通用探针,通过生物素-亲和素系统将探针与分子马达连接构建F0F1-ATPase分子马达检测装置.提取病毒RNA并将其与生物传感器结合的同时启动ATP合成,比较其荧光强度的差别,可以对样品中的RNA进行检测.此方法的病毒RNA检测灵敏度为0.005 ng/mL,对轮状病毒检测特异,与甲肝病毒、诺如病毒无交叉反应,在1h内即可完成检测.运用此方法随机检测15份样品,检测结果与RT-PCR一致.结果表明,分子马达生物传感器检测轮状病毒的方法灵敏、特异,可用于食源性轮状病毒的快速检测.     

产溶剂梭菌分子遗传操作技术研究进展

产溶剂梭菌是一类重要的工业微生物.通过遗传改造以优化产溶剂梭菌的发酵性能一直是溶剂制造技术研究的重要课题,但长期受限于该类菌并不完善的遗传操作工具,未见明显突破.近年来,随着TargeTron基因中断、大片段基因整合等新技术和新方法的出现,其分子遗传改造已取得较大进展.文中对产溶剂梭菌的分子遗传操作工具研究进展进行了总结,并指出了现有技术在效率及全面性方面的不足.基于此,今后应进一步优化现有的梭菌基因失活技术,如建立基于同源重组的基因删除和替换;同时也应发展新的分子操作技术,如基因组多位点共编辑、多拷贝定点和随机整合等.     

独脚金内酯调控植物侧枝发育的分子机制及其与生长素交互作用的研究进展

对独脚金内酯（strigolactones,SLs）调控植物侧枝发育的分子机制及其与生长素相互作用的相关研究结果进行了总结和归纳,在此基础上提出今后的重点研究方向。相关的研究结果显示：在拟南芥[Arabidops~thaliana（Linn．）Heynh．]、豌豆（Pisum sativum Linn．）和水稻（Oryza sativa Linn．）等植物多枝突变体中SLs作为可转导信号参与侧枝发育的分子调控,从这些植物中已克隆获得参与SLs生物合成及信号应答途径的一些基因。作为一种植物激素,SLs在侧枝发育调控网络中与生长素相互作用;腋芽发育与其中生长素的输出密切相关,SLs通过调控芽中生长素的输出间接抑制腋芽发育和侧枝生长,而生长素则在SLs生物合成中起调节作用。