Analysis of the reaction mechanism and substrate specificity of haloalkane dehalogenases by sequential and structural comparisons

Haloalkane dehalogenases catalyse environmentally importantdehalogenation reactions. These microbial enzymes representobjects of interest for protein engineering studies, attemptingto improve their catalytic efficiency or broaden their substratespecificity towards environmental pollutants. This paper presentsthe results of a comparative study of haloalkane dehalogenasesoriginating from different organisms. Protein sequences andthe models of tertiary structures of haloalkane dehalogenaseswere compared to investigate the protein fold, reaction mechanismand substrate specificity of these enzymes. Haloalkane dehalogenasescontain the structural motifs of /ß-hydrolases and epoxidaseswithin their sequences. They contain a catalytic triad withtwo different topological arrangements. The presence of a structurallyconserved oxyanion hole suggests the two-step reaction mechanismpreviously described for haloalkane dehalogenase from Xanthobacterautotrophicus GJ10. The differences in substrate specificityof haloalkane dehalogenases originating from different speciesmight be related to the size and geometry of an active siteand its entrance and the efficiency of the transition stateand halide ion stabilization by active site residues. Structurallyconserved motifs identified within the sequences can be usedfor the design of specific primers for the experimental screeningof haloalkane dehalogenases. Those amino acids which were predictedto be functionally important represent possible targets forfuture site-directed mutagenesis experiments.     

特异性抗肿瘤免疫核糖核酸的提取

用H22细胞接种于小鼠腹腔，抽取癌性腹水，分离肿瘤细胞，用肿瘤细胞裂解物加弗氏完全佐剂充分乳化制备成免疫原，接种于小鼠，取小鼠的免疫器官提取核糖核酸，并进行相关性能检测。结果表明该方法制备提取免疫的核糖核酸携带供体肿瘤特异性免疫信息，能提高抗肿瘤的疗效。     

Preference of certain scolytidae for different conifers

Nine Scolytidae (Cryphalus piceae, Cryphalus abietis, Pityokteines curvidens, Dendroctonus micans, Ips sexdentatus, Ips typographus,Orthotomicus erosus, Tomicus piniperda, andPhloeosinus bicolor) were subjected to olfaction tests on ten conifer species taken two by two. These conifers wereAbies cephalonica, Abies nordmanniana, Picea abies, Picea orientalis, Pinus pinaster, Pinus sylvestris, Pinus brutia, Pinus laricio,Cupressus atlantica, andCupressus sempervirens. A statistical study of the results, by means of the factorial analysis of correspondence completed by the duo preference test, commonly used in sensory analysis, revealed a taxonomic clustering by genus of the plant species and analogous specific attraction behavior for the insects.Pityokteines curvidens has a behavior analogous to that of the twoCryphalus considered.Phloesinus bicolor shows a very strong specificity forCupressus. The essential oils of the conifers were analyzed to determine their terpene composition and the ten odor spectra thus obtained were compared. The hierarchical classification, using a Euclidian distance, brought out similarities in the spectra, especially in the case ofPinus. It is shown that definitive establishment of Scolytidae is not due to the presence in the odor spectrum of any particular terpenoid. The attractive power of a species results from the synergism of the different terpenes. Moreover the definitive establishment of the insects also depends on their sensorial adaptation to volatile substances which can be wider or narrower for the species studied.     

A within-modality test of semantic knowledge: The Size/Weight Attribute Test.

Individuals with semantic dementia (SD; n=12) or Alzheimer's disease (AD; n=20) and healthy volunteers (n=40) were tested on tasks probing attribute rather than associative knowledge of animals and objects in the visual and verbal domains. The tasks were within modality, in that they probed knowledge in a single presentation modality (pictures or written words) and did not require cross-modal matching. Participants were required to make the same simple judgment about triads of animal stimuli ("Which is the largest and smallest?") and object stimuli ("Which is the heaviest and lightest?"). Control participants scored at ceiling on this simple task. Overall, the SD patients were significantly more impaired on this task than were the AD patients, who in turn were significantly more impaired than the controls. There was a strong trend for SD but not AD patients to show worse performance with verbal than visual material. However, no significant effects of category were found in either patient group. The Size/Weight Attribute Test has the potential to assess modality-specific deficits of semantic knowledge. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

派生诉讼中的取证与举证问题研究

派生诉讼中的原告和被告存在严重的信息不对称,这导致原告举证困难.对涉及举证问题的前置请求阶段和审判阶段均从举证责任归属、举证程度要求以及获取证据途径几个方面进行了研究,力求在便利股东提起派生诉讼的同时对公司的经营管理不造成过度的干扰.     

Synthesis and Evaluation of a Nonsteroidal Anti-inflammatory Polymeric Prodrug for Sustained and Site-Specific Delivery

A new polymerizable drug derivative of diclofenac sodium was synthesized and characterized in terms of melting point, elemental analysis, and infrared spectroscopy. It was then polymerized to obtain a new polymeric prodrug. The prodrug was evaluated for its viscosity, drug content, and in vitro drug release behavior at pH 1.2 and 7.2. The in vitro studies showed that the drug release takes place predominantly at the higher pH and in a sustained manner, as hypothesized. Stability at room temperature, bioavailability, and ulcer-inducing effect of the polymeric prodrug were also studied. The investigations showed complete drug absorption from the polymeric prodrug with a statistically significant decrease in ulcer scoring effect, thus showing its potential for site-specific and sustained drug delivery.     

Genistein Inhibition of Topoisomerase II�� Expression Participated by Sp1 and Sp3 in HeLa Cell

Genistein (4′, 5, 7-trihydroxyisoflavone) is an isoflavone compound obtained from plants that has potential applications in cancer therapy. However, the molecular mechanism of the action of genistein on cancer cell apoptosis is not well known. In this study, we investigated the effect of genistein on topoisomerase II-α (Topo IIα), an important protein involved in the processes of DNA replication and cell proliferation. The results revealed that inhibition of Topo IIα expression through the regulation of Specificity protein 1 and Specificity protein 3 may be one of the reasons for genistein’s induction of HeLa cell apoptosis.     

Conversion of a Mono‐ and Diacylglycerol Lipase into a Triacylglycerol Lipase by Protein Engineering

Despite the fact that most lipases are believed to be active against triacylglycerides, there is a small group of lipases that are active only on mono‐ and diacylglycerides. The reason for this difference in substrate scope is not clear. We tried to identify the reasons for this in the lipase from Malassezia globosa. By protein engineering, and with only one mutation, we managed to convert this enzyme into a typical triacylglycerol lipase (the wild‐type lipase does not accept triacylglycerides). The variant Q282L accepts a broad spectrum of triacylglycerides, although the catalytic behavior is altered to some extent. From in silico analysis it seems that specific hydrophobic interactions are key to the altered substrate specificity.     

Comparative analysis of polyspecificity of the endogenous tRNA synthetase of different expression host towards photocrosslinking amino acids using an in silico approach

Photo-induced covalent crosslinking has emerged as the powerful strategy for analyzing and characterizing the protein–protein interaction and mapping protein 3D conformations. In the last decades, a number of photocrosslinking amino acids have been reported but only a few have been efficiently utilized for photocrosslinking purposes. Recently, incorporation of diazirine containing photoactivatable analogs such as photo-methionine, photo-leucine, photo-isoleucine and photo-lysine into target proteins were accomplished in live cells (Human A549cells, HEK 293) by depleting corresponding natural amino acid and supplementing these analogs in the medium. Likewise, incorporation of photo-methionine and photo-leucine is also reported in E. coli. Incorporation of these unnatural amino acids were demonstrated only in a limited number species, thereby conventional methods have been utilized for the protein–protein interaction study in other species. With this in mind, we studied in silico analysis of polyspecificity of four endogenous tRNA synthetases (LeuRS, IleRS, MetRS, and LysRS) from six different species such as Escherichia coli, Pseudomonas fluorescens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Aspergillus oryzae and Homo sapiens towards its photocrosslinking amino acids. In addition, here we describe the active site similarity of different protein bio-factories. Based on the active site similarity and similar binding mode, we predicted that the endogenous tRNA synthetases of all the species are reactive to corresponding photoactivatable analogs. This is the first in silico study to demonstrate that the photocrosslinking unnatural amino acids are recognized by the endogenous tRNA synthetases of different protein expression biofactories.