Where bio meets nano: the many uses for nanoporous aluminum oxide in biotechnology

Porous aluminum oxide (PAO) is a ceramic formed by an anodization process of pure aluminum that enables the controllable assembly of exceptionally dense and regular nanopores in a planar membrane. As a consequence, PAO has a high porosity, nanopores with high aspect ratio, biocompatibility and the potential for high sensitivity imaging and diverse surface modifications. These properties have made this unusual material attractive to a disparate set of applications. This review examines how the structure and properties of PAO connect with its present and potential uses within research and biotechnology. The role of PAO is covered in areas including microbiology, mammalian cell culture, sensitive detection methods, microarrays and other molecular assays, and in creating new nanostructures with further uses within biology.     

Modulation of MPP+ uptake by procyanidins in Caco-2 cells: involvement of oxidation/reduction reactions

It is becoming increasingly evident that the absorption of certain nutrients and drugs and their effects are largely influenced by the concomitant ingestion of other substances. As various xeno- and endobiotics belong to the class of organic cations, the aim of this work was to study the modulation of the intestinal apical uptake of organic cations by diet procyanidins.Five procyanidin fractions with different structural complexity were obtained after fractionation of a grape seed extract. The effect of these compounds on 1-methyl-4-phenylpyridinium (MPP⁺) uptake was evaluated in Caco-2 cells.Apical uptake of ³H-MPP⁺ by Caco-2 cells was increased by a 60 min exposure to 600 μg ml⁻¹ of procyanidin fractions, that increase being positively related with procyanidins structural complexity. It was verified that ³H-MPP⁺ uptake increased with preincubation time. It was speculated that procyanidins were oxidized during preincubation, this change could interfered with transport activity. Tested oxidizing agents showed that the redox state of the transporter could affect its activity. Additionally, trans-stimulation experiments showed that catechin and fraction I (the simpler fraction) can use the same transporter as MPP⁺. The results are compatible with the hypothesis of these compounds being competitive inhibitors of MPP⁺ transport.In conclusion, procyanidins are capable to modulate MPP⁺ apical uptake in Caco-2 cells, this transport being most probably modulated through oxidation-reduction phenomena. Interactions between these compounds and drugs present in the diet may affect their absorption and bioavailability. Both the concentration and complexity of the procyanidin compounds should be taken into account in medical practice.     

Enhancement of arachidonic acid signaling pathway by nicotinic acid receptor HM74A

HM74A is a G protein-coupled receptor for nicotinic acid (niacin), which has been used clinically to treat dyslipidemia for decades. The molecular mechanisms whereby niacin exerts its pleiotropic effects on lipid metabolism remain largely unknown. In addition, the most common side effect in niacin therapy is skin flushing that is caused by prostaglandin release, suggesting that the phospholipase A(2) (PLA(2))/arachidonic acid (AA) pathway is involved. Various eicosanoids have been shown to activate peroxisome-proliferator activated receptors (PPAR) that play a diverse array of roles in lipid metabolism. To further elucidate the potential roles of HM74A in mediating the therapeutic effects and/or side effects of niacin, we sought to explore the signaling events upon HM74A activation. Here we demonstrated that HM74A synergistically enhanced UTP- and bradykinin-mediated AA release in a pertussis toxin-sensitive manner in A431 cells. Activation of HM74A also led to Ca(2+)-mobilization and enhanced bradykinin-promoted Ca(2+)-mobilization through Gi protein. While HM74A increased ERK1/2 activation by the bradykinin receptor, it had no effects on UTP-promoted ERK1/2 activation.Furthermore, UTP- and bradykinin-mediated AA release was significantly decreased in the presence of both MAPK kinase inhibitor PD 098059 and PKC inhibitor GF 109203X. However, the synergistic effects of HM74A were not dramatically affected by co-treatment with both inhibitors, indicating the cross-talk occurred at the receptor level. Finally, stimulation of A431 cells transiently transfected with PPRE-luciferase with AA significantly induced luciferase activity, mimicking the effects of PPARgamma agonist rosiglitazone, suggesting that alteration of AA signaling pathway can regulate gene expression via endogenous PPARs.     

Studying base pair open-close kinetics of tRNALeu by TROSY-based proton exchange NMR spectroscopy

The millisecond conformational flexibility is functionally important for nucleic acids and can be studied through probing the base pair open-close kinetics by proton exchange nuclear magnetic resonance (NMR) spectroscopy. Here, the traditional imino proton exchange NMR experiments were modified with transverse relaxation optimized spectroscopy and were applied to accurately measure imino proton exchange rates of all base pairs in Escherichia coli tRNA^Leu (CAG), and their dependence on magnesium ion concentration. Finally, we correlated millisecond conformational flexibility with aminoacylation of tRNA^Leu and proposed that the flexibility of the acceptor stem and the core region might contribute to aminoacylation of tRNA^Leu.     

Insights into Substrate Specificity of Geranylgeranyl Reductases Revealed by the Structure of Digeranylgeranylglycerophospholipid Reductase, an Essential Enzyme in the Biosynthesis of Archaeal Membrane Lipids

Archaeal membrane lipids consist of branched, saturated hydrocarbons distinct from those found in bacteria and eukaryotes. Digeranylgeranylglycerophospholipid reductase (DGGR) catalyzes the hydrogenation process that converts unsaturated 2,3-di-O-geranylgeranylglyceryl phosphate to saturated 2,3-di-O-phytanylglyceryl phosphate as a critical step in the biosynthesis of archaeal membrane lipids. The saturation of hydrocarbon chains confers the ability to resist hydrolysis and oxidation and helps archaea withstand extreme conditions. DGGR is a member of the geranylgeranyl reductase family that is also widely distributed in bacteria and plants, where the family members are involved in the biosynthesis of photosynthetic pigments. We have determined the crystal structure of DGGR from the thermophilic heterotrophic archaea Thermoplasma acidophilum at 1.6 Å resolution, in complex with flavin adenine dinucleotide (FAD) and a bacterial lipid. The DGGR structure can be assigned to the well-studied, p-hydroxybenzoate hydroxylase (PHBH) SCOP superfamily of flavoproteins that include many aromatic hydroxylases and other enzymes with diverse functions. In the DGGR complex, FAD adopts the IN conformation (closed) previously observed in other PHBH flavoproteins. DGGR contains a large substrate-binding site that extends across the entire ligand-binding domain. Electron density corresponding to a bacterial lipid was found within this cavity. The cavity consists of a large opening that tapers down to two, narrow, curved tunnels that closely mimic the shape of the preferred substrate. We identified a sequence motif, PxxYxWxFP, that defines a specificity pocket in the enzyme and precisely aligns the double bond of the geranyl group with respect to the FAD cofactor, thus providing a structural basis for the substrate specificity of geranylgeranyl reductases. DGGR is likely to share a common mechanism with other PHBH enzymes in which FAD switches between two conformations that correspond to the reductive and oxidative half cycles. The structure provides evidence that substrate binding likely involves conformational changes, which are coupled to the two conformational states of the FAD.     

Improved understanding of the interactions and complexities of biological nitrogen and phosphorus removal processes

Nitrogen and phosphorus removal from wastewater is now considered essential for the protection of our waterways. Biological nutrient removal processes are generally the most efficient and cost-effective solution to achieve this. While the principles of these processes are well known, intriguing and useful details are being discovered with the recent advances in bio-process engineering and microbial sciences. Phosphorus accumulating organisms have only been identified in recent years, and there are now competing glycogen accumulating organisms being found in biological phosphorus removal systems. These can possibly explain the reasons for the variable phosphorus removal performance of certain systems, and their control can help in the development of more stable and better performing processes. Detailed investigations of the traditional nitrification-denitrification systems, but also of novel developments for nitrogen removal, reveal a more complex and diverse range of processes involved in these transformations. Increasingly, linked phosphorus and nitrogen removal processes are being developed, creating further opportunities to optimise the technologies. However, this might also bring certain risks such as the potential to produce the greenhouse-gas nitrous oxide (N₂O) rather than nitrogen gas as the final denitrification product. A range of recent developments in these areas is covered in this paper.     

B3HM细胞免疫小鼠脾细胞scFv噬菌体展示文库的构建及表达

目的: 利用噬菌体展示技术构建B3HM细胞免疫小鼠的脾细胞表达scFv文库。方法: 用人骨髓细胞系B3HM细胞免疫小鼠,取其脾细胞采用RT-PCR方法扩增VH 和Vk基因并克隆入噬菌体展示表达载体,构建scFv文库,测定文库的库容量,BstNI酶切单克隆分析文库的多样性,对文库进行富集检测,鉴定单克隆噬菌体与B3HM细胞结合反应。结果:文库的库容为5×106cfu,单克隆的BstNI酶切图谱显示多样性,单克隆噬菌体抗体与B3HM细胞呈阳性反应。结论:噬菌体展示文库的成功构建为寻找新的致白血病相关基因,阐明白血病发病机理奠定了基础。     

‘Candidatus Competibacter'-lineage genomes retrieved from metagenomes reveal functional metabolic diversity

The glycogen-accumulating organism (GAO) ‘Candidatus Competibacter'' (Competibacter) uses aerobically stored glycogen to enable anaerobic carbon uptake, which is subsequently stored as polyhydroxyalkanoates (PHAs). This biphasic metabolism is key for the Competibacter to survive under the cyclic anaerobic-‘feast'': aerobic-‘famine'' regime of enhanced biological phosphorus removal (EBPR) wastewater treatment systems. As they do not contribute to phosphorus (P) removal, but compete for resources with the polyphosphate-accumulating organisms (PAO), thought responsible for P removal, their proliferation theoretically reduces the EBPR capacity. In this study, two complete genomes from Competibacter were obtained from laboratory-scale enrichment reactors through metagenomics. Phylogenetic analysis identified the two genomes, ‘Candidatus Competibacter denitrificans'' and ‘Candidatus Contendobacter odensis'', as being affiliated with Competibacter-lineage subgroups 1 and 5, respectively. Both have genes for glycogen and PHA cycling and for the metabolism of volatile fatty acids. Marked differences were found in their potential for the Embden–Meyerhof–Parnas and Entner–Doudoroff glycolytic pathways, as well as for denitrification, nitrogen fixation, fermentation, trehalose synthesis and utilisation of glucose and lactate. Genetic comparison of P metabolism pathways with sequenced PAOs revealed the absence of the Pit phosphate transporter in the Competibacter-lineage genomes—identifying a key metabolic difference with the PAO physiology. These genomes are the first from any GAO organism and provide new insights into the complex interaction and niche competition between PAOs and GAOs in EBPR systems.     

PGC-1β和SREBP-1c在猪脂肪细胞分化过程中的相互作用

为研究过氧化物酶体增殖物激活受体γ辅激活因子1β(PGC-1β)与SREBP-1c在猪前体脂肪细胞分化过程中的表达规律及其相互作用,分析二者功能上的联系,采用Western 印迹及细胞免疫荧光技术检测PGC-1β与SREBP-1c在猪脂肪细胞分化过程中的表达,shRNA干扰和免疫共沉淀技术分别探讨了PGC-1β对SREBP-1c的调节作用及2种蛋白质在体内的结合活性.结果显示,PGC-1β与SREBP-1c 蛋白的表达均随猪脂肪细胞分化逐渐增加,且在分化细胞的核和胞浆中均有分布. 干扰PGC-1β显著下调了SREBP-1c和脂肪细胞分化标记基因C/EBPα的表达(P<0.05),同时降低了细胞内甘油三酯的积累.免疫共沉淀证明,PGC-1β与SREBP-1c蛋白在猪脂肪细胞分化过程中存在结合作用. 以上结果表明,PGC-1β能够促进猪脂肪细胞分化并对SREBP-1c有调节和结合作用,推测二者的结合可能与其对脂肪细胞的分化调节机制相关,将对PGC-1β调控脂肪细胞分化的功能和机理研究提供新途径.