Effects of metal ions on activity of plasmin

The effects of some metal ions on amidolytic and fibrinogenolytic activities of highly purified human plasmin were investigated in vitro. In the presence of Zn²⁺, Cu²⁺, Cd²⁺, and Au⁺ in the incubation mixture at the concentrations of 1×10⁻⁵−1×10⁻³ M, the anidolytic plasmin activity was strongly inhibited, whereas Ca²⁺ and Mg²⁺ at the same concentrations were not effective. The analysis of the kinetic study has shown that Zn²⁺ or Cu²⁺ acts as mixed-type inhibitors of plasmin activity. The inhibition of amidolytic plasmin activity by Zn²⁺ and Cu²⁺ was reduced in the presence of EDTA, histidine, or albumin. Incubation of plasmin with Zn²⁺ or Cu²⁺ (at the concentration of 5×10⁻⁴ M) resulted in complete loss of its proteolytic action on fibrinogen, whereas Cd²⁺ and Au⁺ under the same conditions only partially inhibited this process.     

Genome-wide patterns of expression in Drosophila pure species and hybrid males

One of the most fundamental questions for understanding the origin of species is why genes that function to cause fertility in a pure-species genetic background fail to produce fertility in a hybrid genetic background. A related question is why the sex that is most often sterile or inviable in hybrids is the heterogametic (usually male) sex. In this survey, we have examined the extent and nature of differences in gene expression between fertile adult males of two Drosophila species and sterile hybrid males produced from crosses between these species. Using oligonucleotide microarrays and real-time quantitative polymerase chain reaction, we have identified and confirmed that differences in gene expression exist between pure species and hybrid males, and many of these differences are quantitative rather than qualitative. Furthermore, genes that are expressed primarily or exclusively in males, including several involved in spermatogenesis, are disproportionately misexpressed in hybrids, suggesting a possible genetic cause for their sterility.     

具有a-淀粉酶分泌活性以及低双乙酰产量的啤酒酵母工程菌的构建

扣囊复膜酵母(Saccharomycopsis fibuligera)因具有较强的a-淀粉酶以及葡聚糖酶活性, 使其在以淀粉为唯一碳源的培养基上能够良好的生长。从其基因组中克隆了a-淀粉酶的编码区, 构建了由酵母磷酸甘油酸激酶基因(PGK1)启动子、酿酒酵母a-因子信号序列以及扣囊复膜酵母a-淀粉酶基因编码序列组成的基因表达盒。将该表达盒插入到质粒pPLZ-2的ILV2基因序列内部, 使其两翼具有ILV2基因的同源区。将该表达盒通过同源重组的方式整合到啤酒酵母工业菌株YSF-5的a-乙酰乳酸合成酶(AHAS)基因ILV2内部。在以淀粉为唯一碳源的培养基上进行转化子的筛选。通过多对引物PCR、a-淀粉酶活性以及AHAS活性分析对转化子进行鉴定, 得到一株具有a-淀粉酶分泌表达活性、较低AHAS活性, 并且发酵液中双乙酰产量也相对较低的啤酒酵母工程菌。该菌株在非选择压力条件下连续培养50代后仍然保持其遗传稳定性。还对pH、温度以及金属离子对该转化菌株的a-淀粉酶活性的影响进行了研究。由于所构建的菌株不含有非酵母来源的DNA, 所以生物安全性相对较高, 对酵母育种以及啤酒生产工业都具有较为重要的意义。     

The High Throughput Sequence Annotation Service (HT-SAS) – the shortcut from sequence to true Medline words

Background  

Advances in high-throughput technologies available to modern biology have created an increasing flood of experimentally determined facts. Ordering, managing and describing these raw results is the first step which allows facts to become knowledge. Currently there are limited ways to automatically annotate such data, especially utilizing information deposited in published literature.     

Quantitative Proteomics of Presynaptic Mitochondria Reveal an Overexpression and Biological Relevance of Neuronal MitoNEET in Postnatal Brain Development

Although it has been recognized that energy metabolism and mitochondrial structure and functional activity in the immature brain differs from that of the adult, few studies have examined mitochondria specifically at the neuronal synapse during postnatal brain development. In this study, we examined the presynaptic mitochondrial proteome in mice at postnatal day 7 and 42, a period that involves the formation and maturation of synapses. Application of two independent quantitative proteomics approaches – SWATH‐MS and super‐SILAC – revealed a total of 40 proteins as significantly differentially expressed in the presynaptic mitochondria. In addition to elevated levels of proteins known to be involved in ATP metabolic processes, our results identified increased levels of mitoNEET (Cisd1), an iron‐sulfur containing protein that regulates mitochondrial bioenergetics. We found that mitoNEET overexpression plays a cell‐type specific role in ATP synthesis and in neuronal cells promotes ATP generation. The elevated ATP levels in SH‐SY5Y neuroblastoma cells were associated with increased mitochondrial membrane potential and a fragmented mitochondrial network, further supporting a role for mitoNEET as a key regulator of mitochondrial function.     

Kinetics and mechanism of oxygen-independent hydrocarbon hydroxylation by ethylbenzene dehydrogenase

Ethylbenzene dehydrogenase (EBDH) from the denitrifying bacterium Azoarcus sp. strain EbN1 (to be renamed Aromatoleum aromaticum) catalyzes the oxygen-independent, stereospecific hydroxylation of ethylbenzene to (S)-1-phenylethanol, the first known example of direct anaerobic oxidation of a nonactivated hydrocarbon. The enzyme is a trimeric molybdenum/iron-sulfur/heme protein of 155 kDa that is quickly inactivated in air in its reduced state. Enzyme activity can be coupled to ferricenium tetrafluoroborate, providing a convenient way for kinetic measurements. EBDH exhibits activity with a wide range of ethylbenzene analogues, which were analyzed for their kinetic parameters, stoichiometry, and formed products. The reactivity was correlated to the chemical structures by a quantitative structure-activity relationship (QSAR) model. On the basis of these results, quantum chemical calculations of DeltaG298 for formation of carbocations of the respective substrates were performed and used in reactivity analysis. A putative reaction mechanism is proposed on the basis of the experimental results and theoretical considerations. Finally, the enzyme reaction has been established in an electrochemical reactor, allowing sustained enzymatic reaction and potential technical applications of the enzyme.     

Adhesion of the genome-sequenced <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">cremoris</Emphasis> IBB477 strain is mediated by specific molecular determinants

Understanding the nature of mucus-microbe interactions will provide important information that can help to elucidate the mechanisms underlying probiotic adhesion. This study focused on the adhesive properties of the Lactococcus lactis subsp. cremoris IBB477 strain, previously shown to persist in the gastrointestinal tract of germ-free rats. The shear flow-induced detachment of L. lactis cells was investigated under laminar flow conditions. Such a dynamic approach demonstrated increased adhesion to bare and mucin-coated polystyrene for IBB477, compared to that observed for the MG1820 control strain. To identify potential genetic determinants giving adhesive properties to IBB477, the improved high-quality draft genome sequence comprising chromosome and five plasmids was obtained and analysed. The number of putative adhesion proteins was determined on the basis of surface/extracellular localisation and/or the presence of adhesion domains. To identify proteins essential for the IBB477 specific adhesion property, nine deletion mutants in chromosomal genes have been constructed and analysed using adhesion tests on bare polystyrene as well as mucin-, fibronectin- or collagen IV-coated polystyrene plates in comparison to the wild-type strain. These experiments demonstrated that gene AJ89_07570 encoding a protein containing DUF285, MucBP and four Big_3 domains is involved in adhesion to bare and mucin-coated polystyrene. To summarise, in the present work, we characterised the adhesion of IBB477 under laminar flow conditions; identified the putative adherence factors present in IBB477, which is the first L. lactis strain exhibiting adhesive and mucoadhesive properties to be sequenced and demonstrated that one of the proteins containing adhesion domains contributes to adhesion.     

Automatic particle selection: results of a comparative study

Manual selection of single particles in images acquired using cryo-electron microscopy (cryoEM) will become a significant bottleneck when datasets of a hundred thousand or even a million particles are required for structure determination at near atomic resolution. Algorithm development of fully automated particle selection is thus an important research objective in the cryoEM field. A number of research groups are making promising new advances in this area. Evaluation of algorithms using a standard set of cryoEM images is an essential aspect of this algorithm development. With this goal in mind, a particle selection "bakeoff" was included in the program of the Multidisciplinary Workshop on Automatic Particle Selection for cryoEM. Twelve groups participated by submitting the results of testing their own algorithms on a common dataset. The dataset consisted of 82 defocus pairs of high-magnification micrographs, containing keyhole limpet hemocyanin particles, acquired using cryoEM. The results of the bakeoff are presented in this paper along with a summary of the discussion from the workshop. It was agreed that establishing benchmark particles and using bakeoffs to evaluate algorithms are useful in promoting algorithm development for fully automated particle selection, and that the infrastructure set up to support the bakeoff should be maintained and extended to include larger and more varied datasets, and more criteria for future evaluations.     

New surface contacts formed upon reductive lysine methylation: Improving the probability of protein crystallization

Surface lysine methylation (SLM) is a technique for improving the rate of success of protein crystallization by chemically methylating lysine residues. The exact mechanism by which SLM enhances crystallization is still not clear. To study these mechanisms, and to analyze the conditions where SLM will provide the optimal benefits for rescuing failed crystallization experiments, we compared 40 protein structures containing N,N-dimethyl-lysine (dmLys) to a nonredundant set of 18,972 nonmethylated structures from the PDB. By measuring the relative frequency of intermolecular contacts (where contacts are defined as interactions between the residues in proximity with a distance of 3.5 Å or less) of basic residues in the methylated versus nonmethylated sets, dmLys-Glu contacts are seen more frequently than Lys-Glu contacts. Based on observation of the 10 proteins with both native and methylated structures, we propose that the increased rate of contact for dmLys-Glu is due to both a slight increase in the number of amine-carboxyl H-bonds and to the formation of methyl C–H···O interactions. By comparing the relative contact frequencies of dmLys with other residues, the mechanism by which methylation of lysines improves the formation of crystal contacts appears to be similar to that of Lys to Arg mutation. Moreover, analysis of methylated structures with the surface entropy reduction (SER) prediction server suggests that in many cases SLM of predicted SER sites may contribute to improved crystallization. Thus, tools that analyze protein sequences and mark residues for SER mutation may identify proteins with good candidate sites for SLM.