The Emerging Nexus of Active DNA Demethylation and Mitochondrial Oxidative Metabolism in Post-Mitotic Neurons

The variable patterns of DNA methylation in mammals have been linked to a number of physiological processes, including normal embryonic development and disease pathogenesis. Active removal of DNA methylation, which potentially regulates neuronal gene expression both globally and gene specifically, has been recently implicated in neuronal plasticity, learning and memory processes. Model pathways of active DNA demethylation involve ten-eleven translocation (TET) methylcytosine dioxygenases that are dependent on oxidative metabolites. In addition, reactive oxygen species (ROS) and oxidizing agents generate oxidative modifications of DNA bases that can be removed by base excision repair proteins. These potentially link the two processes of active DNA demethylation and mitochondrial oxidative metabolism in post-mitotic neurons. We review the current biochemical understanding of the DNA demethylation process and discuss its potential interaction with oxidative metabolism. We then summarise the emerging roles of both processes and their interaction in neural plasticity and memory formation and the pathophysiology of neurodegeneration. Finally, possible therapeutic approaches for neurodegenerative diseases are proposed, including reprogramming therapy by global DNA demethylation and mitohormesis therapy for locus-specific DNA demethylation in post-mitotic neurons.     

The Uneven Rate of the Molecular Evolution of Gene Sequences of DNA-Dependent RNA Polymerase I of the Genus Lamium L

RNA polymerase type I (plastid-encoded polymerase, PEP) is one of the key chloroplast enzymes. However, the rpo genes that encode its subunits (rpoA, rpoB, rpoC1 and rpoC2) are relatively rapidly evolving sequences. The aim of this study was to investigate the rate of the molecular evolution of rpo genes and to evaluate them as phylogenetic markers on the example of the genus Lamium L. (Lamiaceae). The analyzed genes were shown to differ in the level of variation, rate of intragenic mutations, phylogenetic informativeness, and in the impact of these mutations on the properties of encoded peptides. Destabilizing effects of the positive pressure were observed in all genes examined coding for PEP enzyme. We have demonstrated the relationship between mutations fixed by positive selection and the separation of phylogenetic lines within the genus Lamium. The study showed also that the rpo genes were reliable phylogenetic markers, useful in the reconstruction of interconnections of species belonging to the same genus. Of the four tested genes, the most promising phylogenetic marker was rpoA gene, while the least useful gene appeared to be rpoC1.     

Analysis of the Insect OS-D-Like Gene Family

Insect OS-D-like proteins, also known as chemosensory (CSP) or sensory appendage proteins (SAP), are broadly expressed in various insect tissues, where they are thought to bind short to medium chain length fatty acids and their derivatives. Although their specific function remains uncertain, OS-D-like members have been isolated from sensory organs (including the sensillum lymph in some cases), and a role in olfaction similar to that of the insect odorant binding proteins (OBP) has been suggested for some. We have identified 15 new OS-D-like sequences: four from cDNA clones described herein and 11 from sequence databases. The os-d-like genes from the Anopheles gambiae, Apis mellifera, Drosophila melanogaster, and Drosophila pseudoobscura genomes typically have single, small introns with a conserved splice site. Together with all family members entered on GenBank, a total of 70 OS-D-like proteins, representing the insect orders Diptera, Dictyoptera, Hymenoptera, Lepidoptera, Orthoptera, and Phasmatodea, were analyzed. A neighbor joining distance phenogram identified several protein similarity classes that were characterized by highly conserved sequence motifs, including (A) N-terminal YTTKYDN(V/I)(N/D)(L/V)DEIL, (B) central DGKELKXX(I/L)PDAL, and (C) C-terminal KYDP. In contrast, three similarity classes were characterized by their diversion from these conserved motifs. The functional importance of conserved amino acid residues is discussed in relation to the crystal and NMR structures of MbraCSPA6.     

Molecular Evolution of Aralkylamine N-Acetyltransferase in Fish: A Genomic Survey

All living organisms synchronize biological functions with environmental changes; melatonin plays a vital role in regulating daily and seasonal variations. Due to rhythmic activity of the timezyme aralkylamine N-acetyltransferase (AANAT), the blood level of melatonin increases at night and decreases during daytime. Whereas other vertebrates have a single form of AANAT, bony fishes possess various isoforms of aanat genes, though the reasons are still unclear. Here, we have taken advantage of multiple unpublished teleost aanat sequences to explore and expand our understanding of the molecular evolution of aanat in fish. Our results confirm that two rounds of whole-genome duplication (WGD) led to the existence of three fish isoforms of aanat, i.e., aanat1a, aanat1b, and aanat2; in addition, gene loss led to the absence of some forms from certain special fish species. Furthermore, we suggest the different roles of two aanat1s in amphibious mudskippers, and speculate that the loss of aanat1a, may be related to terrestrial vision change. Several important sites of AANAT proteins and regulatory elements of aanat genes were analyzed for structural comparison and functional forecasting, respectively, which provides insights into the molecular evolution of the differences between AANAT1 and AANAT2.     

低相对分子质量细菌纤维素的生物合成

通过木醋杆菌菌株HN001静态培养,生物合成了低相对分子质量细菌纤维素(LMBC),其培养基是以海南椰子水为原料,添加了糖和其他盐类化合物。研究了影响LMBC产率的几个因素,如培养温度、培养时间和培养基初始pH。获得高产率LMBC的适宜条件是:培养时间72 h,培养温度33℃,培养基初始pH=4,LMBC产率达1.2 g/L。用凝胶过滤色谱仪(GFC)测定了其相对分子质量及其分布。研究了培养基初始pH从3.5到5.5变化对LMBC相对分子质量及其分布的影响,结果表明,该pH范围内相对分子质量的变化不大,其分布指数均约为1.3,说明相对分子质量均匀。用透射电镜测试了其形貌,证明LMBC的形貌近似球形,大小约20 nm;LMBC冷冻干燥后经红外光谱确证了其结构。     

H_5N_1亚型禽流感病毒血凝素基因的克隆及分子进化分析

目的　克隆H5N1 亚型禽流感病毒HA基因并进行分子进化分析。方法　应用RT PCR方法 ,以 2株H5N1 亚型禽流感病毒RNA为模板 ,扩增了HA全基因cDNA。将HAcDNA基因克隆于pUCm T载体 ,并对其进行了测序。结果　所克隆的HA基因全长为 1731bp ,共编码 5 6 8个氨基酸。将该毒株与其它 10株H5亚型禽流感病毒HA基因序列核苷酸及氨基酸进行比较 ,其核苷酸同源性在 80 5 %～ 99 2 %之间 ,氨基酸序列同源性在 89 4 %～98 6 %之间。高致病性毒株HA基因裂解位点存在多个碱性氨基酸插入。结论　为禽流感基因工程疫苗的研制奠定了基础 ,同时通过分子进化分析也揭示了各毒株间的亲缘关系。     

蛋白印迹膜洗脱方式的考察

选用丙烯酰胺作为功能单体,N,N’-亚甲基双丙烯酰胺为交联剂,制备牛血红蛋白的分子印迹聚合物。通过不同方法洗脱印迹膜,比较洗脱后印迹膜的洗脱率、吸附量以及选择性吸附效果。结果表明,酶洗脱的印迹膜具有较高的洗脱率,而通过醋酸和十二烷基硫酸钠洗脱的印迹膜对模板的吸附量和选择性却明显高于其它洗脱方法。     

Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High‐Molecular‐Weight Hyaluronic Acid

Hyaluronic acid (HA), with diverse cosmetic and medical applications, is the natural glycosaminoglycan product of HA synthases. Although process and/or metabolic engineering are used for industrial HA production, the potential of protein engineering has barely been realised. Herein, knowledge‐gaining directed evolution (KnowVolution) was employed to generate an HA synthase variant from Pasteurella multocida (pmHAS) with improved chain‐length specificity and a twofold increase in mass‐based turnover number. Seven improved pmHAS variants out of 1392 generated by error‐prone PCR were identified; eight prospective positions were saturated and the most beneficial amino acid substitutions were recombined. After one round of KnowVolution, the longest HA polymer (<4.7 MDa), through an engineered pmHAS variant in a cell‐free system, was synthesised. Computational studies showed that substitutions from the best variant (T40L, V59M and T104A) are distant from the glycosyltransferase sites and increase the flexibility of the N‐terminal region of pmHAS. Taken together, these findings suggest that the N terminus may be involved in HA synthesis and demonstrate the potential of protein engineering towards improved HA synthase activity.     

CO2在醇中溶解度的测定与分子连接性指数的关联

用恒定溶剂法测定了303.15K下CO2在乙醇、正丙醇、异丙醇、正丁醇、正戊醇、异戊醇、正己醇、正辛醇、乙二醇、1,2-丙二醇中的溶解度。实验表明:随着碳链的增长,吸收CO2的效果越好,CO2在正辛醇中的溶解度最大,CO2在二元醇中的亨利常数显著大于一元醇。利用线性回归方法建立CO2在醇中的亨利常数lgH与分子连接性指数的相关关系模型,模型计算简单易行,对CO2在醇中的亨利常数有良好的估算和预测能力,相关系数R>0.99,计算值和实验值吻合良好。研究表明:用一阶分子连接性指数1χv、羟基的个数N、经验参数COH三个参数可以描述CO2在醇中的溶解特性lgH,且精确度很高。     

α位羰基烯胺酮代间苯二酚的合成和荧光性质研究

合成了一类α位羰基烯胺酮间苯二酚新化合物,通过1H NMR,13C NMR,IR,EA等方法进行了表征,并对8种化合物与五种不同的金属阳离子作用后的荧光光谱和紫外-可见吸收光谱的变化进行了对比研究,发现1-(5-乙酰基-2,4-二羟苯基-3-(4-氯苯胺)丙-2-烯酮化合物)对过渡金属铜离子有明显的特征荧光淬灭识别能力,因此可以作为一种金属离子荧光分子探针选择性识别过渡金属Cu2+离子。     

有机硅交联聚合物的分子模拟

由正硅酸乙酯(TEOS)和甲基三乙氧基硅烷(MTEOS)出发,先构建了聚合物的单体模型,再构建单链,最后通过手动建立化学键的方法构建了有机硅树脂交联体系的网络模型,并且验证了该模型的有效性.通过比较选择了适合有机硅交联体系的非键作用力和聚合度,再通过模拟有机硅交联体系的密度与温度的关系图、比体积与温度的关系图预测了有机硅树脂交联体系的玻璃化转变温度(Tg).     

Study on Immobilized Metallocene and Single-Site Catalysts for the Preparation of Ultra-High Molecular Weight Polyethylene at Various Polymerization Conditions

Ultra-high molecular weight polyethylene (UHMWPE) possesses advantages over conventional polyolefins such as excellent mechanical properties. Recent progresses in transition-metal complexes have led to the discovery of highly active catalysts for the preparation of UHMWPE. In this study, Ti with bis(phenoxy-imine) ligand (FI catalyst) and Me₂Si(C₅Me₄)(N-tBu)TiCl₂ (CGC) were immobilized on silica and tested for the preparation of UHMWPE. Results revealed that soluble FI catalyst and CGC can produce polyethylene having relatively high molecular weight above 10⁶ g/mol, which also can be successfully immobilized on carriers for better adaptability to production processes.     

Use of the MLPA Assay in the Molecular Diagnosis of Gene Copy Number Alterations in Human Genetic Diseases

Multiplex Ligation-dependent Probe Amplification (MLPA) assay is a recently developed technique able to evidence variations in the copy number of several human genes. Due to this ability, MLPA can be used in the molecular diagnosis of several genetic diseases whose pathogenesis is related to the presence of deletions or duplications of specific genes. Moreover, MLPA assay can also be used in the molecular diagnosis of genetic diseases characterized by the presence of abnormal DNA methylation. Due to the large number of genes that can be analyzed by a single technique, MLPA assay represents the gold standard for molecular analysis of all pathologies derived from the presence of gene copy number variation. In this review, the main applications of the MLPA technique for the molecular diagnosis of human diseases are described.     

Human Aquaporin-4 and Molecular Modeling: Historical Perspective and View to the Future

Among the different aquaporins (AQPs), human aquaporin-4 (hAQP4) has attracted the greatest interest in recent years as a new promising therapeutic target. Such a membrane protein is, in fact, involved in a multiple sclerosis-like immunopathology called Neuromyelitis Optica (NMO) and in several disorders resulting from imbalanced water homeostasis such as deafness and cerebral edema. The gap of knowledge in its functioning and dynamics at the atomistic level of detail has hindered the development of rational strategies for designing hAQP4 modulators. The application, lately, of molecular modeling has proved able to fill this gap providing a breeding ground to rationally address compounds targeting hAQP4. In this review, we give an overview of the important advances obtained in this field through the application of Molecular Dynamics (MD) and other complementary modeling techniques. The case studies presented herein are discussed with the aim of providing important clues for computational chemists and biophysicists interested in this field and looking for new challenges.     

A Hamiltonian Replica Exchange Molecular Dynamics (MD) Method for the Study of Folding,Based on the Analysis of the Stabilization Determinants of Proteins

Herein, we present a novel Hamiltonian replica exchange protocol for classical molecular dynamics simulations of protein folding/unfolding. The scheme starts from the analysis of the energy-networks responsible for the stabilization of the folded conformation, by means of the energy-decomposition approach. In this framework, the compact energetic map of the native state is generated by a preliminary short molecular dynamics (MD) simulation of the protein in explicit solvent. This map is simplified by means of an eigenvalue decomposition. The highest components of the eigenvector associated with the lowest eigenvalue indicate which sites, named “hot spots”, are likely to be responsible for the stability and correct folding of the protein. In the Hamiltonian replica exchange protocol, we use modified force-field parameters to treat the interparticle non-bonded potentials of the hot spots within the protein and between protein and solvent atoms, leaving unperturbed those relative to all other residues, as well as solvent-solvent interactions. We show that it is possible to reversibly simulate the folding/unfolding behavior of two test proteins, namely Villin HeadPiece HP35 (35 residues) and Protein A (62 residues), using a limited number of replicas. We next discuss possible implications for the study of folding mechanisms via all atom simulations.