Hetero- and euchromatin of synchronous Euglena cells : I. Physical fractionation of nuclei into differentially condensed chromatin

Several attempts were made to isolate intact nuclei and fractionate condensed and extended chromatin from synchronized cells of Euglena gracilis Z. Different factors affecting the recovery and the intactness of nuclei have been tested: detergent concentration, incubation time and addition of Mn²⁺ (0.13 mM) and/or spermidine (0.14 mM) as protective agents. Interphase and mitotic nuclei show preserved chromatin when Mn²⁺ is included, while the combination of Mn²⁺ and spermidine—and, to a lesser extent, spermidine alone—leads to mitotic nuclei with enhanced clumped chromatin. The common procedure to fractionate Euglena chromatin involves swelling of nuclei before disruption. We proved that this step induces artefactual decondensation of packed heterochromatin. Two alternative methods are compared with separate condensed and dispersed chromatin: (1) breakage of swollen nuclei and subsequent addition of divalent cations and/or spermidine with mild pressure shearing forces; (2) disruption of nuclei in a medium containing Mn²⁺ as a protective agent, without swelling. Electron microscopy study indicates that the normal packed appearance of condensed chromatin is preserved, according to the second procedure, while extensive shearing is necessary. Template capacity of the extended chromatin is significantly higher in both methods. Relative amounts of condensed and dispersed chromatin during interphase and mitosis are discussed.     

Data mining of molecular dynamics trajectories of nucleic acids

Analysis, storage, and transfer of molecular dynamic trajectories are becoming the bottleneck of computer simulations. In this paper we discuss different approaches for data mining and data processing of huge trajectory files generated from molecular dynamic simulations of nucleic acids.     

Involvement of ABC transporters in melanogenesis and the development of multidrug resistance of melanoma

Because melanomas are intrinsically resistant to conventional radiotherapy and chemotherapy, many alternative treatment approaches have been developed such as biochemotherapy and immunotherapy. The most common cause of multidrug resistance (MDR) in human cancers is the expression and function of one or more A TP‐b inding c assette (ABC) transporters that efflux anticancer drugs from cells. Melanoma cells express a group of ABC transporters (such as ABCA9, ABCB1, ABCB5, ABCB8, ABCC1, ABCC2, and ABCD1) that may be associated with the resistance of melanoma cells to a broad range of anticancer drugs and/or of melanocytes to toxic melanin intermediates and metabolites. In this review, we propose a model (termed the ABC‐M model) in which the intrinsic MDR of melanoma cells is at least in part because of the transporter systems that may also play a critical role in reducing the cytotoxicity of the melanogenic pathway in melanocytes. The ABC‐M model suggests molecular strategies to reverse MDR function in the context of the melanogenic pathway, which could open therapeutic avenues towards the ultimate goal of circumventing clinical MDR in patients with melanoma.     

Metal ion interaction with a novel anthracene pendant-armed fluorescent molecular probe. Synthesis, characterization, and fluorescence studies

A new scorpionate system (L) containing an emissive anthracene pendant arm, derived of O¹,O⁷-bis(2-formylphenyl)-1,4,7-trioxaheptane and tren, has been synthesized and characterized. The sensing capability of L towards a range of metal ions has been studied. Protonation and complexation studies, using UV-Vis and fluorescent emission measurements, have been performed with alkaline and alkaline earth metal ions (M = Na(I), K(I), Li(I), Ca(II), Mg(II)), as well as transition and post-transition metal ions (M = Cr(III), Cu(II) and Zn(II), Cd(II), Hg(II) and Al(III)). An increase in the fluorescence emission (CHEF effect) was observed in methanol and in methanol/water mixtures in the presence of Cd(II) (5.0-fold), Zn(II) (4.5-fold), Cr(III) (2.0-fold) and Al(III) (1.8-fold); these results suggest a notable sensing ability of this new N₃O₄ ligand for these metals; these experiments were also performed in the presence of large amounts of alkaline and alkaline earth metal ions.     

Automated genome sequence analysis and annotation.

MOTIVATION: Large-scale genome projects generate a rapidly increasing number of sequences, most of them biochemically uncharacterized. Research in bioinformatics contributes to the development of methods for the computational characterization of these sequences. However, the installation and application of these methods require experience and are time consuming. RESULTS: We present here an automatic system for preliminary functional annotation of protein sequences that has been applied to the analysis of sets of sequences from complete genomes, both to refine overall performance and to make new discoveries comparable to those made by human experts. The GeneQuiz system includes a Web-based browser that allows examination of the evidence leading to an automatic annotation and offers additional information, views of the results, and links to biological databases that complement the automatic analysis. System structure and operating principles concerning the use of multiple sequence databases, underlying sequence analysis tools, lexical analyses of database annotations and decision criteria for functional assignments are detailed. The system makes automatic quality assessments of results based on prior experience with the underlying sequence analysis tools; overall error rates in functional assignment are estimated at 2.5-5% for cases annotated with highest reliability ('clear' cases). Sources of over-interpretation of results are discussed with proposals for improvement. A conservative definition for reporting 'new findings' that takes account of database maturity is presented along with examples of possible kinds of discoveries (new function, family and superfamily) made by the system. System performance in relation to sequence database coverage, database dynamics and database search methods is analysed, demonstrating the inherent advantages of an integrated automatic approach using multiple databases and search methods applied in an objective and repeatable manner. AVAILABILITY: The GeneQuiz system is publicly available for analysis of protein sequences through a Web server at http://www.sander.ebi.ac. uk/gqsrv/submit     

Ca(2+)/Calmodulin-binding proteins from the C. elegans proteome

Calmodulin (CaM) is the primary Ca(2+)-sensor that regulates a wide variety of cellular processes in eukaryotes. Although many Ca(2+)/CaM-binding proteins have been identified, very few such proteins could be found from the genome-wide protein-protein interaction maps of Caenorhabditis elegans constructed by yeast two-hybrid screening. Using a genotype-phenotype conjugation method called mRNA-display, we performed a selection for Ca(2+)/CaM-binding proteins from a proteome library of C. elegans. The method allowed the identification of 9 known and 47 previously uncharacterized Ca(2+)-dependent CaM-binding proteins from the adult worm proteome. The Ca(2+)/CaM-binding properties of these proteins were characterized and their binding motifs were identified. The availability of such information could facilitate our understanding of the signaling pathways mediated by Ca(2+)/CaM in C. elegans. Due to its simplicity and efficiency, the method could be readily applied to examine the Ca(2+)-dependent binding partners of numerous other Ca(2+)-binding proteins, which may play important roles in many signaling pathways in C. elegans.     

Landscape diversity patterns and endemism of Araceae in Ecuador

Araceae is one of the largest herb families in tropical America, but its patterns of diversity and endemism are poorly known. We used predictive distribution modelling in GIS to study Araceae richness on a landscape scale in Ecuador. Modelling was based on georeferenced herbarium collections with humidity and mean annual temperature as climatic variables. Variation partitioning using multiple regression showed that humidity and altitude were main factors in explaining Araceae diversity patterns. Endemism was less well explained by present climatic factors. Unlike common diversity patterns of herbs or epiphytes, Araceae richness was highest in the eastern (Amazon) lowland rain forest with a secondary centre on the Andean foothills of northwestern Ecuador. The peak in endemism was on the western slopes of the Andes, corresponding to areas that have been severely affected by human activities and deforestation. Eastern lowland (Amazonian) forests were poor in endemic Araceae.     

How does the metabolism of tumour cells differ from that of normal cells

Tumour cells thrive in environments that would be hostile to their normal cell counterparts. Survival depends on the selection of cell lines that harbour modifications of both, gene regulation that shifts the balance between the cell cycle and apoptosis and those that involve the plasticity of the metabolic machinery. With regards to metabolism, the selected phenotypes usually display enhanced anaerobic glycolysis even in the presence of oxygen, the so-called Warburg effect, and anabolic pathways that provide precursors for the synthesis of lipids, proteins and DNA. The review will discuss the original ideas of Otto Warburg and how they initially led to the notion that mitochondria of tumour cells were dysfunctional. Data will be presented to show that not only the organelles are viable and respiring, but that they are key players in tumorigenesis and metastasis. Likewise, interconnecting pathways that stand out in the tumour phenotype and that require intact mitochondria such as glutaminolysis will be addressed. Furthermore, comments will be made as to how the peculiarities of the biochemistry of tumour cells renders them amenable to new forms of treatment by highlighting possible targets for inhibitors. In this respect, a case study describing the effect of a metabolite analogue, the alkylating agent 3BP (3-bromopyruvate), on glycolytic enzyme targets will be presented.