Dynamics of Cdc42 network embodies a Turing-type mechanism of yeast cell polarity

Complex biochemical networks can be understood by identifying their principal regulatory motifs and mode of action. We model the early phase of budding yeast cellular polarization and show that the biochemical processes in the presumptive bud site comprise a Turing-type mechanism. The roles of the prototypical activator and substrate are played by GTPase Cdc42 in its active and inactive states, respectively. We demonstrate that the nucleotide cycling of Cdc42 converts cellular energy into a stable cluster of activated Cdc42. This energy drives a continuous membrane-cytoplasmic exchange of the cluster components to counteract diffusive spread of the cluster. This exchange explains why only one bud forms per cell cycle, because the winner-takes-all competition of candidate sites inevitably selects a single site.     

Affixin activates Rac1 via betaPIX in C2C12 myoblast

Affixin/beta-parvin is an integrin-linked kinase (ILK)-binding focal adhesion protein highly expressed in skeletal muscle and heart. To elucidate the possible role of affixin in skeletal muscle, we established stable C2C12 cell line expressing T7-tagged human affixin (C2C12-affixin cells). Exogenous expression of affixin promotes lamellipodium formation where affixin, ILK alphap21-activated kinase (PAK)-interactive exchange factor (PIX) and betaPIX accumulate. The association of affixin and betaPIX was confirmed by immunoprecipitation and pull down assay. In C2C12-affixin cells, an increased level of activated Rac1 but not Cdc42 was observed, and mutant betaPIX lacking guanine nucleotide exchange factor activity inhibited lamellipodium formation. These results suggest that affixin is involved in reorganization of subsarcolemmal cytoskeletal actin by activation of Rac1 through alpha and betaPIXs in skeletal muscle.     

RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors

In the central nervous system, synaptic signal transduction depends on the regulation of neurotransmitter receptors by interacting proteins. Here, we searched for proteins interacting with two metabotropic glutamate receptor type 8 isoforms (mGlu8a and mGlu8b) and identified RanBPM. RanBPM is expressed in several brain regions, including the retina. There, RanBPM is restricted to the inner plexiform layer where it co-localizes with the mGlu8b isoform and processes of cholinergic amacrine cells expressing mGlu2 receptors. RanBPM interacts with mGlu2 and other group II and group III receptors, except mGlu6. Our data suggest that RanBPM might be associated with mGlu receptors at synaptic sites.     

Biological data integration using Semantic Web technologies

Current research in biology heavily depends on the availability and efficient use of information. In order to build new knowledge, various sources of biological data must often be combined. Semantic Web technologies, which provide a common framework allowing data to be shared and reused between applications, can be applied to the management of disseminated biological data. However, due to some specificities of biological data, the application of these technologies to life science constitutes a real challenge. Through a use case of biological data integration, we show in this paper that current Semantic Web technologies start to become mature and can be applied for the development of large applications. However, in order to get the best from these technologies, improvements are needed both at the level of tool performance and knowledge modeling.     

Characterisation of Schiff base and chromophore in green proteorhodopsin by solid-state NMR

The proteorhodopsin family consists of hundreds of homologous retinal containing membrane proteins found in bacteria in the photic zone of the oceans. They are colour tuned to their environment and act as light-driven proton pumps with a potential energetic and regulatory function. Precise structural details are still unknown. Here, the green proteorhodopsin variant has been selected for a chemical shift analysis of retinal and Schiff base by solid-state NMR. Our data show that the chromophore exists in mainly all-trans configuration in the proteorhodopsin ground state. The optical absorption maximum together with retinal and Schiff base chemical shifts indicate a strong interaction network between chromophore and opsin. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mark Lorch and Andreas C. Woerner contributed equally to this work.     

NMR analysis of the closed conformation of syntaxin-1

The Sec1/Munc18 (SM) protein Munc18-1 and the SNAREs syntaxin-1, SNAP-25 and synaptobrevin form the core of the membrane fusion machinery that triggers neurotransmitter release. Munc18-1 binds to syntaxin-1 folded into a closed conformation and to the SNARE complex formed by the three SNAREs, which involves an open syntaxin-1 conformation. The former interaction is likely specialized for neurotransmitter release, whereas SM protein/SNARE complex interactions are likely key for all types of intracellular membrane fusion. It is currently unclear whether the closed conformation is highly or only marginally populated in isolated syntaxin-1, and whether Munc18-1 stabilizes the close conformation or helps to open it to facilitate SNARE complex formation. A detailed NMR analysis now suggests that the closed conformation is almost quantitatively populated in isolated syntaxin-1 in the absence of oligomerization, and indicates that its structure is very similar to that observed previously in the crystal structure of the Munc18-1/syntaxin-1 complex. Moreover, we demonstrate that Munc18-1 binding prevents opening of the syntaxin-1 closed conformation. These results support a model whereby the closed conformation constitutes a key intrinsic property of isolated syntaxin-1 and Munc18-1 binding stabilizes this conformation; in this model, Munc18-1 plays in addition an active role in downstream events after another factor(s) helps to open the syntaxin-1 conformation.     

Influence of proline on the thermostability of the active site and membrane arrangement of transmembrane proteins

Proline residues play a fundamental and subtle role in the dynamics, structure, and function in many membrane proteins. Temperature derivative spectroscopy and differential scanning calorimetry have been used to determine the effect of proline substitution in the structural stability of the active site and transmembrane arrangement of bacteriorhodopsin. We have analyzed the Pro-to-Ala mutation for the helix-embedded prolines Pro⁵⁰, Pro⁹¹, and Pro¹⁸⁶ in the native membrane environment. This information has been complemented with the analysis of the respective crystallographic structures by the FoldX force field. Differential scanning calorimetry allowed us to determine distorted membrane arrangement for P50A and P186A. The protein stability was severely affected for P186A and P91A. In the case of Pro⁹¹, a single point mutation is capable of strongly slowing down the conformational diffusion along the denaturation coordinate, becoming a barrier-free downhill process above 371 K. Temperature derivative spectroscopy, applied for first time to study thermal stability of proteins, has been used to monitor the stability of the active site of bacteriorhodopsin. The mutation of Pro⁹¹ and Pro¹⁸⁶ showed the most striking effects on the retinal binding pocket. These residues are the Pro in closer contact to the active site (activation energies for retinal release of 60.1 and 76.8 kcal/mol, respectively, compared to 115.8 kcal/mol for WT). FoldX analysis of the protein crystal structures indicates that the Pro-to-Ala mutations have both local and long-range effects on the structural stability of residues involved in the architecture of the protein and the active site and in the proton pumping function. Thus, this study provides a complete overview of the substitution effect of helix-embedded prolines in the thermodynamic and dynamic stability of a membrane protein, also related to its structure and function.     

Evaluation of genotoxic effects of Apitol (cymiazole hydrochloride) in vitro by measurement of sister chromatid exchange

Apitol^®, with cymiazole hydrochloride as the active ingredient, is used in bee-keeping against the ectoparasitic mite Varroa destructor. The preparation was evaluated for genotoxicity in cultured human peripheral blood lymphocytes. Sister chromatid exchange, the mitotic index and the cell proliferation index were determined for three experimental concentrations of Apitol^® (0.001, 0.01 and 0.1 mg/ml). All concentrations significantly (p < 0.001) increased the mitotic index (MI = 7.35 ± 0.18%, 8.31 ± 0.20% and 12.33 ± 0.25%, respectively), the proliferative index (PI = 1.83 ± 0.01, 1.84 ± 0.01 and 1.88 ± 0.02, respectively) and the frequency of sister chromatid exchange (SCE = 8.19 ± 1.81, 8.78 ± 1.80 and 13.46 ± 1.88, respectively), suggesting that cymiazole hydrochloride has genotoxic potential.     

以聚乙二醇为层析伴侣同时制备SOD、过氧化氢酶和血红蛋白

发展了一条从红细胞裂解液中同时制备超氧化物歧化酶(SOD)、过氧化氢酶和血红蛋白的新工艺。采用0 75 %的聚乙二醇600作为层析伴侣,使血红蛋白直接流过阴离子交换层析柱,同时吸附SOD和过氧化氢酶。经过梯度洗脱获得SOD和过氧化氢酶组分,再经过疏水性相互作用层析与凝胶过滤层析相串联,使SOD和过氧化氢酶得到纯化。纯化后的SOD和过氧化氢酶的比活力分别达到15932u/mg和65918u/mg ,血红蛋白的纯度达到99.9%以上。总回收率为:SOD ,47.4% ;过氧化氢酶,29.6% ;血红蛋白,88.7%。     

东北老工业基地煤炭矿区环境问题与生态对策

在对我国东北老工业基地煤炭矿区环境问题进行分析的基础上,提出了解决该地区煤炭矿区环境问题的生态对策,包括着重强调煤炭矿区的生态可持续发展对于采矿工业的复兴具有重要推进作用,而澳大利亚关于最佳实践的经验对于今后采煤矿区的发展具有重要借鉴意义。推行生态多元综合治理的土地复垦一修复模式是今后煤炭矿区环境保护的方向。