Synthesis of A Polyaminooligonucleotide Combinatorial Library

Abstract

A new method of synthesis of phosphoramidite of 2′-deoxycytydine carrying a protected spermine moiety at N-4 position is described. A model oligodeoxyribonucleotide containing the specified nucleoside unit has been synthesised. A synthesis of a polyaminooligonucleotide combinatorial library was carried out. The analysis of the above library clearly shows that the presence of spermine moieties in oligodeoxyribonucleotides increases stability of their duplexes.     

Temperature-induced changes of HtrA2(Omi) protease activity and structure

HtrA2(Omi), belonging to the high-temperature requirement A (HtrA) family of stress proteins, is involved in the maintenance of mitochondrial homeostasis and in the stimulation of apoptosis, as well as in cancer and neurodegenerative disorders. The protein comprises a serine protease domain and a postsynaptic density of 95 kDa, disk large, and zonula occludens 1 (PDZ) regulatory domain and functions both as a protease and a chaperone. Based on the crystal structure of the HtrA2 inactive trimer, it has been proposed that PDZ domains restrict substrate access to the protease domain and that during protease activation there is a significant conformational change at the PDZ–protease interface, which removes the inhibitory effect of PDZ from the active site. The crystal structure of the HtrA2 active form is not available yet. HtrA2 activity markedly increases with temperature. To understand the molecular basis of this increase in activity, we monitored the temperature-induced structural changes using a set of single-Trp HtrA2 mutants with Trps located at the PDZ–protease interface. The accessibility of each Trp to aqueous medium was assessed by fluorescence quenching, and these results, in combination with mean fluorescence lifetimes and wavelength emission maxima, indicate that upon an increase in temperature the HtrA2 structure relaxes, the PDZ–protease interface becomes more exposed to the solvent, and significant conformational changes involving both domains occur at and above 30 °C. This conclusion correlates well with temperature-dependent changes of HtrA2 proteolytic activity and the effect of amino acid substitutions (V226K and R432L) located at the domain interface, on HtrA2 activity. Our results experimentally support the model of HtrA2 activation and provide an insight into the mechanism of temperature-induced changes in HtrA2 structure.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-012-0355-1) contains supplementary material, which is available to authorized users.     

Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems

Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers.     

The interplay between inflorescence development and function as the crucible of architectural diversity

Background

Most angiosperms present flowers in inflorescences, which play roles in reproduction, primarily related to pollination, beyond those served by individual flowers alone. An inflorescence''s overall reproductive contribution depends primarily on the three-dimensional arrangement of the floral canopy and its dynamics during its flowering period. These features depend in turn on characteristics of the underlying branching structure (scaffold) that supports and supplies water and nutrients to the floral canopy. This scaffold is produced by developmental algorithms that are genetically specified and hormonally mediated. Thus, the extensive inflorescence diversity evident among angiosperms evolves through changes in the developmental programmes that specify scaffold characteristics, which in turn modify canopy features that promote reproductive performance in a particular pollination and mating environment. Nevertheless, developmental and ecological aspects of inflorescences have typically been studied independently, limiting comprehensive understanding of the relations between inflorescence form, reproductive function, and evolution.

Scope

This review fosters an integrated perspective on inflorescences by summarizing aspects of their development and pollination function that enable and guide inflorescence evolution and diversification.

Conclusions

The architecture of flowering inflorescences comprises three related components: topology (branching patterns, flower number), geometry (phyllotaxis, internode and pedicel lengths, three-dimensional flower arrangement) and phenology (flower opening rate and longevity, dichogamy). Genetic and developmental evidence reveals that these components are largely subject to quantitative control. Consequently, inflorescence evolution proceeds along a multidimensional continuum. Nevertheless, some combinations of topology, geometry and phenology are represented more commonly than others, because they serve reproductive function particularly effectively. For wind-pollinated species, these combinations often represent compromise solutions to the conflicting physical influences on pollen removal, transport and deposition. For animal-pollinated species, dominant selective influences include the conflicting benefits of large displays for attracting pollinators and of small displays for limiting among-flower self-pollination. The variety of architectural components that comprise inflorescences enable diverse resolutions of these conflicts.     

New species of the genus Epistephium (Orchidaceae, Vanilloideae)

The morphological study of the herbarium material representing Epistephium (Orchidaceae, Vanilloideae) led to the discovery of two groups of specimens that significantly differ from all known species of the genus. The results of literature data study and of comparative analysis of those and other specimens suggest that these collections represent new taxa that we describe as E. garayi and E. kubiyuense. The distinguishing features of the species are indicated. As both new species are reported from Colombia (E. garayi, also from Guyana), the key for the determination of all Colombian representatives of the genus is included. Information on the ecology and distribution of newly described taxa is presented.     

Structural analysis of a putative aminoglycoside N-acetyltransferase from Bacillus anthracis

For the last decade, worldwide efforts for the treatment of anthrax infection have focused on developing effective vaccines. Patients that are already infected are still treated traditionally using different types of standard antimicrobial agents. The most popular are antibiotics such as tetracyclines and fluoroquinolones. While aminoglycosides appear to be less effective antimicrobial agents than other antibiotics, synthetic aminoglycosides have been shown to act as potent inhibitors of anthrax lethal factor and may have potential application as antitoxins. Here, we present a structural analysis of the BA2930 protein, a putative aminoglycoside acetyltransferase, which may be a component of the bacterium's aminoglycoside resistance mechanism. The determined structures revealed details of a fold characteristic only for one other protein structure in the Protein Data Bank, namely, YokD from Bacillus subtilis. Both BA2930 and YokD are members of the Antibiotic_NAT superfamily (PF02522). Sequential and structural analyses showed that residues conserved throughout the Antibiotic_NAT superfamily are responsible for the binding of the cofactor acetyl coenzyme A. The interaction of BA2930 with cofactors was characterized by both crystallographic and binding studies.     

(2-Phenylindol-3-yl)methylene]propanedinitriles inhibit the growth of breast cancer cells by cell cycle arrest in G(2)/M phase and apoptosis

Cell cycle arrest of malignant cells is an important option for cancer treatment. In this study, we modified the structure of antimitotic 2-phenylindole-3-carbaldehydes by condensation with malononitrile. The resulting methylene propanedinitriles inhibited the growth of MDA-MB 231 and MCF-7 breast cancer cells with IC(50) values below 100 nM. Though they exhibited similar structure-activity relationships as the aldehydes, they did not inhibit tubulin polymerization but were capable of blocking the cell cycle in G(2)/M phase. The cell cycle arrest was accompanied by apoptosis as demonstrated by the activation of caspases 3 and 9. Since the new 2-phenylindole derivatives also inhibited the growth of transplanted MXT mouse mammary tumors, they are interesting candidates for further development.     

Life cycle thinking in small and medium enterprises: the results of research on the implementation of life cycle tools in Polish SMEs—part 2: LCA related aspects

Purpose

This article is the second part of a series of articles presenting the results of research on the implementation of lifecycle management tools in small- and medium-sized companies in Poland. This work is part of a project financed by the Polish Agency for Enterprise Development (PAED), which began in February 2011. It was carried out by the Wielkopolska Quality Institute, a business environment institution associated with the Polish Centre for life cycle assessment (PCLCA). The main practical objective of the project was to support small and medium enterprises (SMEs) in their business development, e.g. by expanding their horizons beyond the sphere of their operation and identifying new areas for the improvement and promotion of the products and services they offer. The specific objective of the analysis on the environmental impact was an attempt to answer the question of whether environmental LCA is a good management tool for this type of business. Part 2 describes results of the evaluation of the implementation of LCA in SMEs conducted in 46 companies involved in the project.

Methods

In order to assess the effectiveness of the project and the effectiveness of the implementation of LCA and life cycle costing (LCC), a survey was conducted of small and medium businesses where the implementation work had been fully completed. In total, 46 organisations agreed to participate in the LCA survey, which was almost 66 % of all the companies where the LCA and LCC studies had been carried out within the project. The survey was conducted using individual in-depth interviews. Questions to the representatives of the companies referred both to aspects of their functioning in the market (characteristics of a company, its market share, management systems, environmental policy, suppliers and clients) and the operation of their environmental service (assessment of its effectiveness, motivation and difficulties in its implementation), as well as opinions on the potential applications of LCA in their current operations.

Results and discussion

The experience and observations of LCA experts resulting from their cooperation with the organisations analysed are largely supported by the results of the survey. The overall impression gained from the project is that the small- and medium-sized enterprises analysed have a problem with accepting and understanding the life cycle perspective and show limited interest in taking liability for environmental aspects beyond the mandatory legal standards and boundaries of their business operations. The survey shows that the companies rarely analyse environmental aspects appearing on many different stages of the life cycle of their products. Most of them focus on their current operations while trying to meet the mandatory legal requirements relating to environmental protection. It should be noted, however, that SMEs taking part in the studies appreciate the opportunities offered by LCA, their usefulness in business practice, recognise the potential for using life cycle techniques in the future and their impact on the management process, procedure or thinking about the products they manufacture. The result of the study is the identification of four key areas relevant to SMEs which may affect their willingness to adopt the life cycle perspective and undertake environmental measures.

Conclusions

It seems that implementing LCT in small- and medium-sized enterprises requires a special approach. These are often companies with limited human resources (often just a few people) and financial resources (often operating on the verge of survival), with a weak position in a supply chain and, therefore, having various priorities in their daily operation. The researchers also encountered awareness barriers as a result of which the idea of going beyond an organisation and making an entire LCA of a product was often simply misunderstood. The studies conducted among SMEs have shown that managers' own intuition and research on customer preferences were largely conducive to improve existing or introducing new products or services, while changes were mostly introduced due to the requirements of the market, or the desire to reduce costs. It can be assumed that their non-obligatory nature also contributed to the relatively low interest in LCA initiatives and not recognising their usefulness. It seems that it would be useful to carry out relatively simple, but integrated, LCA/LCC analyses in SMEs so that the companies would clearly see the economic effect of the proposed environmental improvements. The analyses conducted lead to the conclusion that the incentive for SMEs to take measures should come from outside, e.g. as requirements for green public procurements, or as part of assessment made by suppliers in a supply chain.