Cellular localization of a Hsp90 homologue in Porphyromonas gingivalis

We previously reported an association between elevated serum antibody titers to the 90-kDa human heat shock protein (Hsp90), periodontal health and colonization by Porphyromonas gingivalis. In this study, we examined the cellular localization of the Hsp90 homologue of P. gingivalis. Cultures of P. gingivalis were heat-stressed (45 degrees C) and examined for localization of the Hsp90 homologue. Heat stress induced a 4-5-fold increase in anti-Hsp90 antibody reactivity over that of the unstressed controls. Western blot analysis revealed two bands (44 and 68 kDa) that reacted with anti-Hsp90 antibodies. The 68-kDa band was heat-inducible, while the 44-kDa band was not. Immunogold staining revealed that the Hsp90 homologue localized principally to the membrane and extracellular vesicles. Subcellular fractionation confirmed that the Hsp90 homologue was primarily membrane-associated.     

Identification of lipophilic flavones and flavonols by comparative HPLC,TLC and UV spectral analysis

The identification of lipophilic flavones and flavonols using a combination of high performance liquid chromatography, thin layer chromatography and UV spectral analysis is discussed. Data are provided for the flavones, apigenin, luteolin and tricetin and twelve of their methyl ethers, 8-hydroxyluteolin, 6-hydroxyluteolin and scutellarein and fourteen of their methyl ethers, and some 6,8-dihydroxyapigenin and 6,8-dihydroxyluteolin derivatives. Data for some forty two flavonols with extra 6- and/or 8-hydroxylation, mostly 6-hydroxykaempferol and quercetagetin derivatives, are also presented. The remaining compounds analysed include fourteen 5-deoxyflavones, four 5-methoxyflavones and five 5-deoxyflavonols plus further 5-hydroxylated flavones and flavonols without B-ring oxidation or with 2'-, 5'- or 6'-hydroxylation.     

Analysis of the stability of cytochrome c(6) with an improved stopped-flow protocol

This work presents an improved stopped-flow protocol for the simultaneous measurement of thermodynamic and kinetic protein stability data from a single experiment, along with a formalism for the global analysis of the data. The method was applied to the comparison of the stabilities of cytochrome c(6) from Anabaena sp. PCC 7119 and one of its mutants (D72K). Compared to the wild type the mutant was found to have a significantly reduced thermodynamic (deltadeltaG(U0)=2.7 kJ mol(-1)) and kinetic stability, as well as a more pronounced shift in transition state structure upon destabilization.     

食线虫真菌侵染性胞外酶和生防应用

食线虫真菌作为重要的植物寄生线虫的生物防治资源,深入了解它们的侵染方式、毒力因子是了解食线虫真菌侵染的分子机理和开发高效、稳定的生物杀线虫制剂的关键。目前的研究表明,食线虫真菌能分泌具有降解线虫体壁或线虫卵壳的胞外酶,它们在食线虫真菌侵染线虫的过程中起着非常重要的作用。对这些侵染性胞外水解酶的深入研究将促进人们对食线虫真菌的侵染过程和侵染机制的了解以及高效生防制剂的开发。综述了近年来食线虫真菌侵染性胞外酶的研究概况,对食线虫真菌胞外丝氨酸蛋白酶进行同源性分析,对以后食线虫真菌侵染性胞外酶的研究和高效生防制剂开发进行了评述。     

Geographic variation in genital morphology of Ciulfina praying mantids

Geographic variation in morphological traits is widespread and important to our current understanding of evolutionary processes. Although male genitalia are perhaps the most divergent morphological traits in animals, geographic variation in genital traits has received little attention and the mechanism driving such variation is unclear. The species isolation hypothesis of genital evolution makes explicit predictions about geographic variation in genitalia predicting patterns of genital divergence that reflect the risk of mating with related but incompatible species. The sexual selection and pleiotropy hypotheses, however, predict general levels of geographic variation that reflect divergent sexual selection pressures or genetic drift. To test these predictions, we investigated geographic variation in genital morphology in the praying mantid genus Ciulfina (Mantodea: Liturgusidae) using elliptic Fourier analysis. We found significant levels of geographic variation in the genital morphology of four Ciulfina species irrespective of the relative proximity of different populations to contact zones with other species. These results reject the species isolation hypothesis, and instead support either the sexual selection or pleiotropy hypotheses to explain patterns of genital evolution in this genus.     

The Use of EGFR Exon 19 and 21 Unlabeled DNA Probes to Screen for Activating Mutations in Non–Small Cell Lung Cancer

Activating mutations in epidermal growth factor receptor-1 (EGFR) are found in 10–15% of Caucasian patients with non–small cell lung carcinoma (NSCLC). Approximately 90% of the mutations are deletions of several amino acids in exon 19 or point mutations in exon 21. Some studies suggest that these mutations identify patients that might benefit from targeted EGFR inhibitor therapy. DNA melting analysis of polymerase chain reaction products can screen for these mutations to identify this patient population. However, amplicon DNA melting analysis, although easily capable of detecting heterozygous mutations by heterodimer formation, becomes more difficult if mutations are homozygous or if the mutant allele is selectively amplified over wild type. Amplification of EGFR is common in NSCLC and this could compromise mutation detection by amplicon melting analysis. To overcome this potential limitation, we developed unlabeled, single-stranded DNA probes, complimentary to EGFR exon 19 and exon 21 where the common activating mutations occur. The unlabeled probes are incorporated into a standard polymerase chain reaction during the amplification of EGFR exons 19 and 21. The probe melting peak is easily distinguished from the amplicon melting peak, and probe melting is altered if mutations are present. This allows for easy identification of activating mutations even in homozygous or amplified states and is useful in the screening of NSCLC for the common EGFR activating mutations.     

A survey of unresolved problems in life cycle assessment

Background, aims, and scope  Life cycle assessment (LCA) stands as the pre-eminent tool for estimating environmental effects caused by products and processes from ‘cradle to grave’ or ‘cradle to cradle.’ It exists in multiple forms, claims a growing list of practitioners, and remains a focus of continuing research. Despite its popularity and codification by organizations such as the International Organization for Standards and the Society of Environmental Toxicology and Chemistry, life cycle assessment is a tool in need of improvement. Multiple authors have written about its individual problems, but a unified treatment of the subject is lacking. The following literature survey gathers and explains issues, problems and problematic decisions currently limiting LCA’s goal and scope definition and life cycle inventory phases. Main features  The review identifies 15 major problem areas and organizes them by the LCA phases in which each appears. This part of the review focuses on the first 7 of these problems occurring during the goal and scope definition and life cycle inventory phases. It is meant as a concise summary for practitioners interested in methodological limitations which might degrade the accuracy of their assessments. For new researchers, it provides an overview of pertinent problem areas toward which they might wish to direct their research efforts. Results and discussion  Multiple problems occur in each of LCA’s four phases and reduce the accuracy of this tool. Considering problem severity and the adequacy of current solutions, six of the 15 discussed problems are of paramount importance. In LCA’s first two phases, functional unit definition, boundary selection, and allocation are critical problems requiring particular attention. Conclusions and recommendations  Problems encountered during goal and scope definition arise from decisions about inclusion and exclusion while those in inventory analysis involve flows and transformations. Foundational decisions about the basis of comparison (functional unit), bounds of the study, and physical relationships between included processes largely dictate the representativeness and, therefore, the value of an LCA. It is for this reason that problems in functional unit definition, boundary selection, and allocation are the most critical examined in the first part of this review.

Non-invasive analysis of cell cycle dynamics in single living cells with Raman micro-spectroscopy

Raman micro-spectroscopy is a laser-based technique which enables rapid and non-invasive biochemical analysis of cells and tissues without the need for labels, markers or stains. Previous characterization of the mammalian cell cycle using Raman micro-spectroscopy involved the analysis of suspensions of viable cells and individual fixed and/or dried cells. Cell suspensions do not provide cell-specific information, and fixing/drying can introduce artefacts which distort Raman spectra, potentially obscuring both qualitative and quantitative analytical results. In this article, we present Raman spectral characterization of biochemical changes related to cell cycle dynamics within single living cells in vitro. Raman spectra of human osteosarcoma cells synchronized in G(0)/G(1), S, and G(2)/M phases of the cell cycle were obtained and multivariate statistics applied to analyze the changes in cell spectra as a function of cell cycle phase. Principal components analysis identified spectral differences between cells in different phases, indicating a decrease in relative cellular lipid contribution to Raman spectral signatures from G(0)/G(1) to G(2)/M, with a concurrent relative increase in signal from nucleic acids and proteins. Supervised linear discriminant analysis of spectra was used to classify cells according to cell cycle phase, and exhibited 97% discrimination between G(0)/G(1)-phase cells and G(2)/M-phase cells. The non-invasive analysis of live cell cycle dynamics with Raman micro-spectroscopy demonstrates the potential of this approach to monitoring biochemical cellular reactions and processes in live cells in the absence of fixatives or labels.     

Models of Monoamine Oxidase A and B Active Sites Obtained by using 3D QSAR with CoMFA Analysis

Abstract

The monoamine oxidase catalyses the oxidative deamination of neuroactive amines. This enzyme exists in two forms A and B, which differ by substrates preference and inhibitors specificity. Investigation of the structures of these enzymes and design new selective inhibitors are of greatly interesting since MAO A inhibitors are used in therapeutic practice as antidepressants and MAO B inhibitors – in the treatment Parkinson's diseases. The three dimension structures of monoamine oxidases are still unknown. Therefore, one of the most perspective approach to define significant features of structure active site is method based on analysis of structure-activity relationship (3D QSAR) with comparison of molecular fields analysis (CoMFA) allowing to get the spatial distribution of important properties affecting the activity.

In present study we investigate the structures of active sites MAO A and B using 16 pyrazinocarbazole derivatives in variant conformation. Majority of pyrazinocarbazole derivatives have a rigit conformation, but three of those is sufficiently flexible. The latters can be in two conformation types: long molecules (substitution accommodate along axis of main structure) and short molecules (substitution accommodate at acute angle about of main structure). Several 3D QSAR and CoMFA models of MAO A and B active sites were design for data sets containing various types of flexible molecules conformation. All obtained models are statistical reliable and have sufficient predictive power for tested compound tetrindole. The best MAO A model that include two flexible molecules in long conformations was obtained, and the longest one of those in short conformation. In contrast, for MAO B model containing all flexible molecules in the short conformations is more preferred.

On the basis of obtained data the schematic models of MAO A and B active sites structures are proposed. According to these models MAO A active site have the narrow long cavity that accommodate long molecules, while MAO B active site is broader and shorter.