Neuritin Activates Insulin Receptor Pathway to Up-regulate Kv4.2-mediated Transient Outward K+ Current in Rat Cerebellar Granule Neurons

Neuritin is a new neurotrophic factor discovered in a screen to identify genes involved in activity-dependent synaptic plasticity. Neuritin also plays multiple roles in the process of neural development and synaptic plasticity. The receptors for binding neuritin and its downstream signaling effectors, however, remain unclear. Here, we report that neuritin specifically increases the densities of transient outward K⁺ currents (I_A) in rat cerebellar granule neurons (CGNs) in a time- and concentration-dependent manner. Neuritin-induced amplification of I_A is mediated by increased mRNA and protein expression of Kv4.2, the main α-subunit of I_A. Exposure of CGNs to neuritin markedly induces phosphorylation of ERK (pERK), Akt (pAkt), and mammalian target of rapamycin (pmTOR). Neuritin-induced I_A and increased expression of Kv4.2 are attenuated by ERK, Akt, or mTOR inhibitors. Unexpectedly, pharmacological blockade of insulin receptor, but not the insulin-like growth factor 1 receptor, abrogates the effect of neuritin on I_A amplification and Kv4.2 induction. Indeed, neuritin activates downstream signaling effectors of the insulin receptor in CGNs and HeLa. Our data reveal, for the first time, an unanticipated role of the insulin receptor in previously unrecognized neuritin-mediated signaling.     

Expression, purification, and characterization of alanine racemase from Pseudomonas putida YZ-26

Alanine racemase catalyzes the interconversion of d- and l-alanine and plays an important role in supplying d-alanine, a component of peptidoglycan biosynthesis, to most bacteria. Alanine racemase exists mostly in prokaryotes and is generally absent in higher eukaryotes; this makes it an attractive target for the design of new antibacterial drugs. Here, we present the cloning and characterization of a new gene-encoding alanine racemase from Pseudomonas putida YZ-26. An open reading frame (ORF) of 1,230 bp, encoding a protein of 410 amino acids with a calculated molecular weight of 44,217.3 Da, was cloned into modified vector pET32M to form the recombinant plasmid pET–alr. After introduction into E.coli BL21, the strain pET-alr/E.coli BL21 expressed His₆-tagged alanine racemase. The recombinant alanine racemase was efficiently purified to homogeneity using Ni²⁺–NTA and a gel filtration column, with 82.5% activity recovery. The amino acid sequence deduced from the alanine racemase gene revealed identity similarities of 97.0, 93, 23, and 22.0% with from P. putida F1, P. putida200, P. aeruginosa, and Salmonella typhimurium, respectively. The recombinant alanine racemase is a monomeric protein with a molecular mass of 43 kDa. The enzyme exhibited activity with l-alanine and l-isoleucine, and showed higher specificity for the former compared with the latter. The enzyme was stable from pH 7.0–11.0; its optimum pH was at 9.0. The optimum temperature for the enzyme was 37°C, and its activity was rapidly lost at temperatures above 40°C. Divalent metals, including Sr²⁺, Mn²⁺, Co²⁺, and Ni²⁺ obviously enhanced enzymatic activity, while the Cu²⁺ ion showed inhibitory effects.     

High-throughput comparison of gene fitness among related bacteria

ABSTRACT: BACKGROUND: The contribution of a gene to the fitness of a bacterium can be assayed by whether and to what degree the bacterium tolerates transposon insertions in that gene. We use this fact to compare the fitness of syntenic homologous genes among related Salmonella strains to reveal differences not apparent at the gene sequence level. RESULTS: A transposon Tn5 derivative was used to construct mutants in Salmonella Typhimurium ATCC14028 (STM1) and Salmonella Typhi Ty2 (STY1), which were then grown in rich media. The locations of 234,152 and 53,556 integration sites, respectively, were mapped by sequencing. These data were compared to similar data available for a different Ty2 strain (STY2) and essential genes identified in E. coli K-12 (ECO). Of 277 genes considered essential in ECO, all had syntenic homologs in STM1, STY1, and STY2, and all but nine genes were either devoid of Tn insertions or had very few. For three of these nine genes, part of the annotated gene lacked Tn integrations (yejM, ftsN and murB). At least one of the other six genes, trpS, had a potentially functionally redundant gene encoded elsewhere in Salmonella but not in ECO. An additional 165 genes were almost entirely devoid of transposon integrations in all three Salmonella strains examined, including many genes associated with protein and DNA synthesis. Four of these genes (STM14_1498.L, STM14_2872, STM14_3360.RJ, and STM14_5442) are not found in E. coli. Notable differences in the extent of gene selection were also observed among the three different Salmonella isolates. Mutations in hns, for example, were selected against in STM1 but not in the two STY strains, which have a defect in rpoS rendering hns nonessential. CONCLUSIONS: Comparisons among transposon integration profiles from different members of a species and among related species, all grown in similar conditions, identify differences in gene fitness among syntenic homologous genes. Further differences in fitness profiles among shared genes can be expected in other selective environments, with potential relevance for comparative systems biology.     

金沙江干热河谷锥连栎群落物种组成与多样性特征

金沙江干热河谷元谋段残存的锥连栎群落中植物种类组成较为简单,共发现68种植物,隶属于35科60属,其中,禾本科、蝶形花科、菊科、唇形科等科植物占有较大优势;从植物生活型和功能型来看,群落内以草本植物和多年生植物数量居多,分别占所有植物种类的58.8%和63.2%。不同类型群落中,除了扭黄茅始终为各群落中最为重要的优势种之外,同一草本植物在不同群落中的作用和地位存在较大差异;群落物种多样性及相似性程度较为低下,其Shannon-Weiner指数在1.7～2.6之间,且与群落受干扰程度存在较大关联。     

普萘洛尔对映异构体诱导HUVEC细胞的蛋白质表达谱差异

手性药物只能通过严格的手性识别才能选择性地与特定生物大分子相互作用,在药动学、药效学等方面上表现出手性特征.以非选择性β肾上腺素能受体阻滞剂普萘洛尔（PRO）的对映异构体R(+)/S(-)-PRO为模型药物,分别作用于人脐静脉内皮细胞（HUVEC）,提取全细胞蛋白质,经双向电泳、MALDI-TOF-MS、SWISSPROT数据库分析鉴定差异表达蛋白质;共筛选出22个差异表达蛋白质点,鉴定了HSP86、HSP84、GRP75、KLC18、KBTB2、TGM2、GBLP、GCNT2、RAB36、KLH34等10种蛋白质.研究表明,PRO对映异构体可引起广泛的基因表达改变,涉及信号分子、代谢酶、骨架蛋白、伴侣蛋白等,且具有显著的手性特征,这可能与PRO显著的手性生物学特征有紧密联系,但仍需开展进一步深入研究,以明确产生PRO手性生物学特征的多种途径和机制.蛋白质组学技术为深入了解药物的手性生物学特征及其作用机制提供了新的思路和策略,对手性药物开发和临床合理用药有着重要的意义.     

极端嗜热古菌———芝田硫化叶菌DNA 连接酶的生化性质

极端嗜热古菌———芝田硫化叶菌 DNA 连接酶 (Ssh 连接酶 ) 的最适辅因子为 ATP ,在 dATP 存在时,该酶也能表现出较弱的连接活性 . ATP 或 dATP 都能够使该酶发生腺苷化,腺苷化的 Ssh 连接酶能够将腺苷基团转移至含切刻的 DNA 上 . 电泳迁移率改变实验表明, Ssh 连接酶能够结合双链 DNA ,且与含切刻及不含切刻的 DNA 结合的亲和力相同,但不结合单链 DNA. 酵母双杂交实验显示,硫磺矿硫化叶菌 ( 与芝田硫化叶菌亲缘关系很近 ) 的 DNA 连接酶,与该菌所含的 3 个增殖细胞核抗原 (PCNA) 同源蛋白中的一个 (PCNA-1) 有相互作用,而与另外 2 个同源蛋白 (PCNA-like 和 PCNA-2) 则无相互作用 . 在古菌中高度保守的 Sac10b 蛋白家族成员 Ssh10b 能够激活 Ssh 连接酶的活性,而硫化叶菌中的主要染色体蛋白——— 7 ku DNA 结合蛋白 (Ssh7) 则对该酶活性没有影响 .     

Delivery of Therapeutic AGT shRNA by PEG-Bu for Hypertension Therapy

Gene silencing by RNA interference (RNAi) is a promising approach for gene therapy. However, up to today, it is still a major challenge to find safe and efficient non-viral vectors. Previously, we reported PEI-Bu, a small molecular weight PEI derivative, as an efficient non-viral carrier. However, like many PEI-based polymers, PEI-Bu was too toxic. In order to reduce cytotoxicity while maintain or even enhance transfecion efficiency, we modified PEI-Bu with poly(ethylene glycol) (PEG) to obtain PEG-Bu, and used it to delivery a theraputic short hairpin RNA (shRNA) targeting angiotensinogen (AGT) to normal rat liver cells (BRL-3A), which was a key target for the treatment of hypertension. The structure of PEG-Bu was confirmed by proton nuclear magnetic resonance (¹H-NMR). Gel permeation chromatography (GPC) showed that the weight average molecular weight (Mw) of PEG-Bu was 5880 Da, with a polydispersity of 1.58. PEG-Bu could condense gene cargo into spherical and uniform nanoparticles with particle size (65–88 nm) and zeta potential (7.3–9.6 mV). Interestingly and importantly, PEG-Bu displayed lower cytotoxicity and enhanced tranfection efficiency than PEI-Bu after PEGylation in both normal cells BRL-3A and tumor cells HeLa. Moreover, PEG-Bu could efficiently delivery AGT shRNA to knockdown the AGT expression. To sum up, PEG-Bu would be a promising non-viral vector for delivering AGT shRNA to BRL-3A cells for hypertension therapy.