MAR-FISH技术及其在环境微生物群落与功能研究中的应用

对复杂环境中微生物群落结构和功能的研究是微生物生态学的重要任务。尽管现代分子生物学技术已经成功地用于解析环境中微生物的群落结构, 但是这些方法并不能提供微生物的原位生理学信息。而一种新的方法, 微观放射自显影和荧光原位杂交集成技术(MAR-FISH)则能够同时在单细胞水平上, 检测复杂环境中微生物的系统发育信息及其生理特性。本文总结了MAR-FISH方法的原理, 实验步骤及其在环境微生物群落与功能研究中的应用。     

Single cell and neural process experimentation using laterally applied electrical fields between pairs of closely apposed microelectrodes with vertical sidewalls

As biomedical research has moved increasingly towards experimentation on single cells and subcellular structures, there has been a need for microscale devices that can perform manipulation and stimulation at a correspondingly small scale. We propose a microelectrode array (MEA) featuring thickened microelectrodes with vertical sidewalls (VSW) to focus electrical fields horizontally on targets positioned in between paired electrodes. These microelectrodes were fabricated using gold electroplating that was molded by photolithographically patterned SU-8 photoresist. Finite element modeling showed that paired VSW electrodes produce more uniform electrical fields compared to conventional planar microelectrodes. Using paired microelectrodes, 3 μm thick and spaced 10 μm apart, we were able to perform local electroporation of individual axonal processes, as demonstrated by entry of EGTA to locally chelate intra-axonal calcium, quenching the fluorescence of a pre-loaded calcium indicator dye. The same electrode configuration was used to electroporate individual cells, resulting in the targeted transfection of a transgene expressing a cytoplasmically soluble green fluorescent protein (GFP). In addition to electropration, our electrode configuration was also capable of precisely targeted field stimulation on individual neurons, resulting in action potentials that could be tracked by optical means. With its ability to deliver well-characterized electrical fields and its versatility, our configuration of paired VSW electrodes may provide the basis for a new tool for high-throughput and high-content experimentation in broad areas of neuroscience and biomedical research.     

一株高抗汞细菌的分离鉴定及其抗性基因的克隆与表达

摘要：【目的】本研究旨在从重金属汞抗性细菌中分离鉴定汞抗性基因【方法】从北京凉水河河床底泥中分离抗汞细菌,采用16S rRNA基因序列分析结合生理生化特征对菌株进行鉴定。根据GenBank中已发表的多种抗汞细菌的merA基因序列设计引物,以抗性细菌基因组DNA为模板,扩增merA基因,并在大肠杆菌（Escherichia coli）BL21(DE3)表达。同时对表达菌株的重金属汞抗性进行测定。【结果】分离得到一株能在含HgCl2为70 mg/L的平板上生长良好的高抗汞细菌,编号为KHg2。16S rRNA     

Mechanism and significance of specific proteolytic cleavage of Reelin

Reelin is a secreted glycoprotein essential for normal brain development and function. In the extracellular milieu, Reelin is subject to specific cleavage at two (N-t and C-t) sites. The N-t cleavage of Reelin is implicated in psychiatric and Alzheimer’s diseases, but the molecular mechanism and physiological significance of this cleavage are not completely understood. Particularly, whether the N-t cleavage affects the signaling activity of Reelin remains controversial.Here, we show that the protease in charge of the N-t cleavage of Reelin requires the activity of certain proprotein convertase family for maturation and has strong affinity for heparin. By taking advantage of these observations, we for the first time succeeded in obtaining “Uncleaved” and “Completely Cleaved” Reelin proteins. The N-t cleavage splits Reelin into two distinct fragments and virtually abolishes its signaling activity. These findings provide an important biochemical basis for the function of Reelin proteolysis in brain development and function.     

Phosphorylated and ubiquitinated TDP-43 pathological inclusions in ALS and FTLD-U are recapitulated in SH-SY5Y cells

We report phosphorylated and ubiquitinated aggregates of TAR DNA binding protein of 43 kDa (TDP-43) in SH-SY5Y cells similar to those in brains of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). Two candidate sequences for the nuclear localization signal were examined. Deletion of residues 78-84 resulted in cytoplasmic localization of TDP-43, whereas the mutant lacking residues 187-192 localized in nuclei, forming unique dot-like structures. Proteasome inhibition caused these to assemble into phosphorylated and ubiquitinated TDP-43 aggregates. The deletion mutants lacked the exon skipping activity of cystic fibrosis transmembrane conductance regulator (CFTR) exon 9. Our results suggest that intracellular localization of TDP-43 and proteasomal function may be involved in inclusion formation and neurodegeneration in TDP-43 proteinopathies.     

Syntheses, structures and antimicrobial activities of various metal complexes of hinokitiol

Metal-oxygen bonding complexes (M = Mg^II, Mn^II, Ni^II, Mo^VI, W^VI, Pd^II, Sb^III, Bi^III, Fe^III, Ti^IV, K^I, Ba^II, Zr^IV and Hf^IV) with a hinokitiol (Hhino; 2-hydroxy-4-isopropylcyclohepta-2,4,6-trienone or β-thujaplicin) ligand, which has two unequivalent oxygen donor atoms, were synthesized and characterized by elemental analysis, TG/DTA, FT-IR and solution (¹H and ¹³C) NMR spectroscopy. Single-crystal X-ray structure analysis revealed various molecular structures for the complexes, which were classified into several families of family, i.e. type A [M^II(hino)₂(L)]₂ (M = Mg^II, Mn^II, Ni^II; L = EtOH or MeOH), with a dimeric structure consisting of one bridging hino⁻ anion, one chelating hino⁻ anion and one alcohol or water molecule, type B, with the octahedral, cis-dioxo, bis-chelate complexes cis-[M^VIO₂(hino)₂] (M = Mo^VI, W^VI), type C, with square planar complex [M^II(hino)₂] (M = Pd^II), type D, with tris-chelate, 7-coordinate complexes with one inert electron pair [M^III(hino)₃] (M = Sb^III, Bi^III), type D′, with the bis-chelate, pseudo-6-coordinate complexes with one inert electron pair [M^III(hino)₂X] (M = Sb^III, X = Br), type E, with tris-chelate, 6-coordinate complexes with Δ and Λ isomers [M^III(hino)₃] (M = Fe^III), type E′ of bis-chelate, 6-coordinate complex [M^IV(hino)₂X₂] (M = Ti^IV, X = Cl), type F, with water-soluble alkali metal salts [M^I(hino)] (M = K^I), and type H, with tetrakis-chelate, 8-coordinate complexes [M^IV(hino)₄](M = Zr^IV, Hf^IV). These structural features were compared with those of metal complexes with a related ligand, tropolone (Htrop). The antimicrobial activities of these complexes, evaluated in terms of minimum inhibitory concentration (MIC; μg mL⁻¹) in two systems, were compared to elucidate the relationship between structure and antimicrobial activity.     

The Calcium-Dependent and Calcium-Independent Membrane Binding of Synaptotagmin 1: Two Modes of C2B Binding

The Ca²⁺-independent membrane interactions of the soluble C2 domains from synaptotagmin 1 (syt1) were characterized using a combination of site-directed spin labeling and vesicle sedimentation. The second C2 domain of syt1, C2B, binds to membranes containing phosphatidylserine and phosphatidylcholine in a Ca²⁺-independent manner with a lipid partition coefficient of approximately 3.0 × 10² M^− 1. A soluble fragment containing the first and second C2 domains of syt1, C2A and C2B, has a similar affinity, but C2A alone has no detectable affinity to phosphatidylcholine/phosphatidylserine bilayers in the absence of Ca²⁺. Although the Ca²⁺-independent membrane affinity of C2B is modest, it indicates that this domain will never be free in solution within the cell. Site-directed spin labeling was used to obtain bilayer depth restraints, and a simulated annealing routine was used to generate a model for the membrane docking of C2B in the absence of Ca²⁺. In this model, the polybasic strand of C2B forms the membrane binding surface for the domain; however, this face of C2B does not penetrate the bilayer but is localized within the aqueous double layer when C2B is bound. This double-layer location indicates that C2B interacts in a purely electrostatic manner with the bilayer interface. In the presence of Ca²⁺, the membrane affinity of C2B is increased approximately 20-fold, and the domain rotates so that the Ca²⁺-binding loops of C2B insert into the bilayer. This Ca²⁺-triggered conformational change may act as a switch to modulate the accessibility of the polybasic face of C2B and control interactions of syt1 with other components of the fusion machinery.     

Fas Apoptosis Inhibitory Molecule Contains a Novel β-Sandwich in Contact with a Partially Ordered Domain

Fas apoptosis inhibitory molecule (FAIM) is a soluble cytosolic protein inhibitor of programmed cell death and is found in organisms throughout the animal kingdom. A short isoform of FAIM is expressed in all tissue types, while an alternatively spliced long isoform is specifically expressed in the brain. Here, the short isoform is shown to consist of two independently folding domains in contact with each other. The NMR solution structure of the C-terminal domain of murine FAIM is solved in isolation and revealed to be a novel protein fold, a noninterleaved seven-stranded β-sandwich. The structure and sequence reveal several residues that are likely to be involved in functionally significant interactions with the N-terminal domain or other binding partners. Chemical shift perturbation is used to elucidate contacts made between the N-terminal domain and the C-terminal domain.     

The ATPase Cycle of PcrA Helicase and Its Coupling to Translocation on DNA

The superfamily 1 bacterial helicase PcrA has a role in the replication of certain plasmids, acting with the initiator protein (RepD) that binds to and nicks the double-stranded origin of replication. PcrA also translocates single-stranded DNA with discrete steps of one base per ATP hydrolyzed. Individual rate constants have been determined for the DNA helicase PcrA ATPase cycle when bound to either single-stranded DNA or a double-stranded DNA junction that also has RepD bound. The fluorescent ATP analogue 2′(3′)-O-(N-methylanthraniloyl)ATP was used throughout all experiments to provide a complete ATPase cycle for a single nucleotide species. Fluorescence intensity and anisotropy stopped-flow measurements were used to determine rate constants for binding and release. Quenched-flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor MDCC-PBP was used to measure phosphate release kinetics. The chemical cleavage step is the rate-limiting step in the cycle and is essentially irreversible and would result in the bound ATP complex being a major component at steady state. This cleavage step is greatly accelerated by bound DNA, producing the high activation of this protein compared to the protein alone. The data suggest the possibility that ADP is released in two steps, which would result in bound ADP also being a major intermediate, with bound ADP·P_i being a very small component. It therefore seems likely that the major transition in structure occurs during the cleavage step, rather than P_i release. ATP rebinding could then cause reversal of this structural transition. The kinetic mechanism of the PcrA ATPase cycle is very little changed by potential binding to RepD, supporting the idea that RepD increases the processivity of PcrA by increasing affinity to DNA rather than affecting the enzymatic properties per se.     

Isoform-Selective Interaction of the Adaptor Protein Tks5/FISH with Sos1 and Dynamins

The adaptor protein Tks5/FISH (tyrosine kinase substrate 5/five SH3 domains, hereafter termed Tks5) is a crucial component of a protein network that controls the invasiveness of cancer cells and progression of Alzheimer's disease. Tks5 consists of an amino-terminal PX domain that is followed by five SH3 domains (SH3A-E), and two different splice variants are expressed. We identified son of sevenless-1 (Sos1) as a novel binding partner of Tks5 and found colocalization of Tks5 with Sos1 in human epithelial lung carcinoma (A549) cells and in podosomes of Src-transformed NIH 3T3 cells. We observe synergistic binding of SH3A and SH3B to Sos1 when peptide arrays are used, indicating that the tandem SH3A and SH3B domains of Tks5 can potentially bind in a superSH3 binding mode, as was described for the homologous protein p47phox. These results are further corroborated by pull-down assays and isothermal titration calorimetry showing that both intact SH3 domains are required for efficient binding to the entire proline-rich domain of Sos1. The presence of a basic insertion between the SH3A and SH3B domains in the long splice variant of Tks5 decreases the affinity to Sos1 isoforms about 10-fold as determined by analytical ultracentrifugation. Furthermore, it leads to an alteration in the recognition of binding motifs for the interaction with Sos1: While the insertion abrogates the interaction with the majority of peptides derived from the proline-rich domains of Sos1 and dynamin that are recognized by the short splice isoform, it enables binding to a different set of peptides including a sequence comprising the splice insertion in the long isoform of Sos1 (Sos1_2). In the absence of the basic insertion, Tks5 was found to bind a range of Sos1 and dynamin peptides including conventional proline-rich motifs and atypical recognition sequences. Hereby, the tandem SH3 domains in Tks5 employ two distinct types of binding modes: One class of peptides is recognized by single SH3 domains, whereas a second class of peptides requires the presence of both domains to bind synergistically. We conclude that the tandem SH3A and SH3B domains of Tks5 constitute a versatile module for the implementation of isoform-specific protein-protein interactions.