Modification of activity and specificity of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 by engineering of its entrance tunnel

Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis; the residue partially blocks the entrance tunnel, and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, 15 active protein variants were obtained, suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterized by determination of specific activities with 12 halogenated substrates and steady-state kinetic parameters with two substrates. The effect of mutation on the enzyme activities varied dramatically with the structure of the substrates, suggesting that extrapolation of one substrate to another may be misleading and that a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and nonpolar amino acid in position 177. This result is consistent with the phylogenetic analysis showing that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants.     

Biochemical function of female-lethal (2)D/Wilms' tumor suppressor-1-associated proteins in alternative pre-mRNA splicing

Genetic and molecular data have implicated the Drosophila gene female-lethal (2)d (fl (2)d) in alternative splicing regulation of genes involved in sexual determination. Sex-specific splicing is under the control of the female-specific regulatory protein sex-lethal (SXL). Co-immunoprecipitation and mass spectrometry results indicate that SXL and FL (2)D form a complex and that the protein VIRILIZER and a Ran-binding protein implicated in protein nuclear import are also present in complexes containing FL (2)D. A human homolog of FL (2)D was identified and cloned. Interestingly, this gene encodes a protein (WTAP) that was previously found to interact with the Wilms' tumor suppressor-1 (WT1), an isoform of which binds to and co-localizes with splicing factors. Alternative splicing of transformer pre-mRNA, a target of SXL regulation, was affected by immunodepletion of hFL (2)D/WTAP from HeLa nuclear extracts, thus arguing for a biochemical function of FL (2)D/WTAP proteins in splicing regulation.     

TableView: portable genomic data visualization

TableView is a generalized scientific visualization program for exploration of various biological data, including EST, SAGE, microarray and annotation data. Written in Java, TableView is portable, is easily used together with other software including DBMSs and is versatile enough to be applied to any tabular data AVAILABILITY: TableView is freely available at: http://ccgb.umn.edu/software/java/apps/TableView/.     

The magnetofection method: using magnetic force to enhance gene delivery

In order to enhance and target gene delivery we have previously established a novel method, termed magnetofection, which uses magnetic force acting on gene vectors that are associated with magnetic particles. Here we review the benefits, the mechanism and the potential of the method with regard to overcoming physical limitations to gene delivery. Magnetic particle chemistry and physics are discussed, followed by a detailed presentation of vector formulation and optimization work. While magnetofection does not necessarily improve the overall performance of any given standard gene transfer method in vitro, its major potential lies in the extraordinarily rapid and efficient transfection at low vector doses and the possibility of remotely controlled vector targeting in vivo.     

Imprinting and disease

Deregulation of imprinted genes has been observed in a number of human diseases such as Beckwith-Wiedemann syndrome, Prader-Willi/Angelman syndromes and cancer. Imprinting diseases are characterised by complex patterns of mutations and associated phenotypes affecting pre- and postnatal growth and neurological functions. Regulation of imprinted gene expression is mediated by allele-specific epigenetic modifications of DNA and chromatin. These modifications preferentially affect central regulatory elements that control in cis over long distances allele-specific expression of several neighbouring genes. Investigations of imprinting diseases have a strong impact on biomedical research and provide interesting models for function and mechanisms of epigenetic gene control.     

Molecular phylogenetics employing modern and ancient DNA

Comparative studies of DNA in recent populations and characterisation of ancient hereditary material have contributed very interesting facts to our understanding of evolution of modern mankind. Analysis of DNA homology in related species, assessment of mutations and polymorphisms in various populations and new DNA sequence data from prehistoric finds allowed - via sophisticated DNA extraction techniques, PCR, sequencing and digitalised processing of genetic information - insights into possible roots of Homo sapiens and related species, migration patterns and ancient cultural habits, thus enrhing the palaeoanthropological discipline. However, a presentation of this development would not be complete without pointing towards the methodological limitations and manifold presentations burdened with artifacts, data misinterpretation and unjustified conclusions. Presently, this modern field of research is in its consolidation phase and new parameters for quality control and authentication are being implemented to avoid spectacular but unfounded reports. It is expected that most of the problems connected to old biomolecules may be closely related to fossilisation parameters. The future challenge will be the full understanding of the complex and multi-faceted processes underlying diagenesis, including the elucidation of nucleic acid postmortem damage".     

Polo-like kinase 1 regulates Nlp,a centrosome protein involved in microtubule nucleation

In animal cells, most microtubules are nucleated at centrosomes. At the onset of mitosis, centrosomes undergo a structural reorganization, termed maturation, which leads to increased microtubule nucleation activity. Centrosome maturation is regulated by several kinases, including Polo-like kinase 1 (Plk1). Here, we identify a centrosomal Plk1 substrate, termed Nlp (ninein-like protein), whose properties suggest an important role in microtubule organization. Nlp interacts with two components of the gamma-tubulin ring complex and stimulates microtubule nucleation. Plk1 phosphorylates Nlp and disrupts both its centrosome association and its gamma-tubulin interaction. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites severely disturbs mitotic spindle formation. We propose that Nlp plays an important role in microtubule organization during interphase, and that the activation of Plk1 at the onset of mitosis triggers the displacement of Nlp from the centrosome, allowing the establishment of a mitotic scaffold with enhanced microtubule nucleation activity.     

Selective staining of proteins with hydrophobic surface sites on a native electrophoretic gel

Chemical proteomics aims to characterize all of the proteins in the proteome with respect to their function, which is associated with their interaction with other molecules. We propose the identification of a subproteomic library of expressed proteins whose native structures are typified by the presence of hydrophobic surface sites, which are often involved in interactions with small molecules, membrane lipids, and other proteins, pertaining to their functions. We demonstrate that soluble globular proteins with hydrophobic surface sites can be detected selectively by staining on an electrophoretic gel run under nondenaturing conditions. The application of these staining techniques may help elucidate new catalytic, transport, and regulatory functionalities in complex proteomic screenings.     

Expression of the human RNA-binding protein HuR in Trypanosoma brucei increases the abundance of mRNAs containing AU-rich regulatory elements

The salivarian trypanosome Trypanosoma brucei infects mammals and is transmitted by tsetse flies. The mammalian ‘bloodstream form’ trypanosome has a variant surface glycoprotein coat and relies on glycolysis while the procyclic form from tsetse flies has EP protein on the surface and has a more developed mitochondrion. We show here that the mRNA for the procyclic-specific cytosolic phosphoglycerate kinase PGKB, like that for EP proteins, contains a regulatory AU-rich element (ARE) that destabilises the mRNA in bloodstream forms. The human HuR protein binds to, and stabilises, mammalian mRNAs containing AREs. Expression of HuR in bloodstream-form trypanosomes resulted in growth arrest and in stabilisation of the EP, PGKB and pyruvate, phosphate dikinase mRNAs, while three bloodstream-specific mRNAs were reduced in abundance. The synthesis and abundance of unregulated mRNAs and proteins were unaffected. Our results suggest that regulation of mRNA stability by AREs arose early in eukaryotic evolution.     

Staphylococcal alpha-toxin provokes neutrophil-dependent cardiac dysfunction: role of ICAM-1 and cys-leukotrienes

The role of polymorphonuclear neutrophils (PMN) in septic myocardial dysfunction is presently unknown. Staphylococcus aureus infections are frequently associated with septic sequelae. Therefore, we perfused isolated rat hearts with low doses of alpha-toxin, the major staphylococcal exotoxin, followed by application of human PMN, N-formyl-methionyl-leucyl-phenylalanine, and arachidonic acid. In contrast to sham-perfused hearts (no alpha-toxin), a rise in coronary perfusion pressure (CPP) and a reduction of contractile function were noted, and cardiac expression of intercellular adhesion molecule (ICAM)-1 was detected by immunohistochemical methods and real-time PCR. Histological analysis and myeloperoxidase activity indicated cardiac PMN accumulation in alpha-toxin-challenged hearts. Major quantities of cysteinyl (cys)-leukotrienes (LT), LTB4, and 5-hydroxyeicosatetraenoic acid (5-HETE) were found in the perfusate of alpha-toxin-exposed hearts. With an anti-ICAM-1 antibody, neutrophil accumulation, leukotriene (LT) synthesis, coronary vasoconstriction, and the accompanying cardiodepression were suppressed. Similarly, the lipoxygenase inhibitor MK-886 blocked LT synthesis and maintained cardiac function. We conclude that low-dose alpha-toxin provokes coronary endothelial ICAM-1 expression and neutrophil accumulation, with subsequent synthesis of cys-LTs, LTB4, and 5-HETE under conditions of appropriate stimulation. This response is linked with coronary vasoconstriction and contractile dysfunction, with cys-LT synthesis and maldistribution of perfusion offered as likely underlying mechanisms.