Biochemistry and Pharmacology - Research Publications
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ItemStructural and biochemical characterization of a mitochondrial peroxiredoxin from Plasmodium falciparum(WILEY, 2006-08)Plasmodium falciparum possesses a single mitochondrion with a functional electron transport chain. During respiration, reactive oxygen species are generated that need to be removed to protect the organelle from oxidative damage. In the absence of catalase and glutathione peroxidase, the parasites rely primarily on peroxiredoxin-linked systems for protection. We have analysed the biochemical and structural features of the mitochondrial peroxiredoxin and thioredoxin of P. falciparum. The mitochondrial localization of both proteins was confirmed by expressing green fluorescent protein fusions in parasite erythrocytic stages. Recombinant protein was kinetically characterized using the cytosolic and the mitochondrial thioredoxin (PfTrx1 and PfTrx2 respectively). The peroxiredoxin clearly preferred PfTrx2 to PfTrx1 as a reducing partner, reflected by the KM values of 11.6 microM and 130.4 microM respectively. Substitution of the two dyads asparagine-62/tyrosine-63 and phenylalanine-139/alanine-140 residues by aspartate-phenylalaine and valine-serine, respectively, reduced the KM for Trx1 but had no effect on the KM of Trx2 suggesting some role for these residues in the discrimination between the two substrates. Solution studies suggest that the protein exists primarily in a homodecameric form. The crystal structure of the mitochondrial peroxiredoxin reveals a fold typical of the 2-Cys class peroxiredoxins and a dimeric form with an intermolecular disulphide bridge between Cys67 and Cys187. These results show that the mitochondrial peroxiredoxin of P. falciparum occurs in both dimeric and decameric forms when purified under non-reducing conditions.
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ItemNo Preview AvailableCharacterization of the nodulation plasmid encoded chemoreceptor gene mcpG from Rhizobium leguminosarum.(Springer Science and Business Media LLC, 2003-01-28)BACKGROUND: In general, chemotaxis in Rhizobium has not been well characterized. Methyl accepting chemotaxis proteins are sensory proteins important in chemotaxis of numerous bacteria, but their involvement in Rhizobium chemotaxis is unclear and merits further investigation. RESULTS: A putative methyl accepting chemotaxis protein gene (mcpG) of Rhizobium leguminosarum VF39SM was isolated and characterized. The gene was found to reside on the nodulation plasmid, pRleVF39d. The predicted mcpG ORF displayed motifs common to known methyl-accepting chemotaxis proteins, such as two transmembrane domains and high homology to the conserved methylation and signaling domains of well-characterized MCPs. Phenotypic analysis of mcpG mutants using swarm plates did not identify ligands for this putative receptor. Additionally, gene knockouts of mcpG did not affect a mutant strain's ability to compete for nodulation with the wild type. Notably, mcpG was found to be plasmid-encoded in all strains of R. leguminosarum and R. etli examined, though it was found on the nodulation plasmid only in a minority of strains. CONCLUSIONS: Based on sequence homology R. leguminosarum mcpG gene codes for a methyl accepting chemotaxis protein. The gene is plasmid localized in numerous Rhizobium spp. Although localized to the sym plasmid of VF39SM mcpG does not appear to participate in early nodulation events. A ligand for McpG remains to be found. Apparent McpG orthologs appear in a diverse range of proteobacteria. Identification and characterization of mcpG adds to the family of mcp genes already identified in this organism.
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ItemNo Preview AvailableRNA interference as a therapeutic strategy for treating CNS disorders.(Elsevier BV, 2006)RNA interference (RNAi) controls gene silencing in most living organisms. The potential clinical applications of RNAi represent a strategy with unsurpassed selectivity, with the ability to target multiple disease-related genes, independent of their perceived drugability. The design of highly selective and efficacious small interfering (siRNAs) and short hairpin RNAs (shRNAs) has become routine, owing to significant progress in modeling and chemistry. RNAi significantly downregulates gene expression and function both in vitro and in vivo, including in the brain. This essay highlights recent findings and how the pharmaceutical industry intends to apply RNAi for the treatment neuropsychiatric and other diseases.
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ItemNo Preview AvailablePET tracer development--a tale of mice and men.(E-MED LTD, 2006-10-31)PET scanning is an emerging technology for the clinical evaluation of many disease processes in man. The vast majority of clinical positron emission tomography (PET) studies are performed using a single tracer, fluorodeoxyglucose. Despite the excellent diagnostic performance of this tracer, it has recognised limitations. New tracers offer the potential to both address these limitations, and to establish new applications for PET. Small animal PET is a logical technique for validating new tracers relevant to human diseases. However, interspecies differences in the handling of chemicals may significantly influence the handling of novel tracers. This requires caution in extrapolating findings in animals to expectations of performance in man. Already there are several examples where biodistribution studies in mice would not have predicted the clinical utility of existing PET tracers. Nevertheless, application of a systematic approach to tracer development is likely to speed transition of new tracers from animals into man.
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ItemNo Preview AvailableAkt in the pathogenesis of COPD.(Informa UK Limited, 2006)In this review we consider the therapeutic potential of targeting Akt for the treatment of COPD. Akt is a serine/threonine protein kinase that functions as a signaling intermediate linked to multiple signaling programs involved in survival, inflammation, and growth. Akt is closely associated with key membrane-bound receptors and represents a convergent integration point for multiple stimuli implicated in COPD pathogenesis. Persistent activation of Akt secondary to somatic mutations in regulatory oncogenes, such as PTEN, may explain why inflammation in COPD does not resolve when smoking is ceased. Akt is also implicated in the systemic manifestations of COPD such as skeletal muscle wasting and metabolic disturbances. Furthermore, targeting Akt may provide a useful means of limiting the severity and duration of disease exacerbations in COPD. As such, Akt represents a particularly attractive therapeutic target for the treatment of COPD. Interestingly, current knowledge suggests that both inhibitors and activators of Akt may be useful for treating different clinical subpopulations of COPD patients.
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ItemNo Preview AvailableWhat is the contribution of respiratory viruses and lung proteases to airway remodelling in asthma and chronic obstructive pulmonary disease?(ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2006)It is well known that the lungs of asthmatics show airway wall remodelling and that asthma exacerbations are linked to respiratory infections. There is some evidence that respiratory infections in early childhood may increase the risk of developing asthma later in life. Chronic obstructive pulmonary disease (COPD), by definition, involves structural changes to the airways. However, very little is known about what role virus infections play in the development of this remodelling. This review considers the role of matrix metalloproteases and neutrophil elastase in remodelling, and whether the induction of proteases and other mediators during respiratory virus infections may contribute to the development of airway remodelling.
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ItemProspects for the treatment of drug-resistant malaria parasites(FUTURE MEDICINE LTD, 2006-06)Widespread parasitic resistance has led to an urgent need for the development and implementation of new drugs for the treatment of Plasmodium falciparum malaria. Artemisinin and its derivatives are becoming increasingly important, used preferably in combination with a second antimalarial agent to increase the efficacy and slow the development of resistance. However, cost, production and pharmacological issues associated with artemisinin derivatives and potential partner drugs are hindering the implementation of combination therapies. This article reviews the molecular basis of the action of, and resistance to, different antimalarials and examines the prospects for the next generation of drugs to combat this potentially lethal human pathogen.
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ItemModuleFinder and CoReg: alternative tools for linking gene expression modules with promoter sequences motifs to uncover gene regulation mechanisms in plants(BMC, 2006)BACKGROUND: Uncovering the key sequence elements in gene promoters that regulate the expression of plant genomes is a huge task that will require a series of complementary methods for prediction, substantial innovations in experimental validation and a much greater understanding of the role of combinatorial control in the regulation of plant gene expression. RESULTS: To add to this larger process and to provide alternatives to existing prediction methods, we have developed several tools in the statistical package R. ModuleFinder identifies sets of genes and treatments that we have found to form valuable sets for analysis of the mechanisms underlying gene co-expression. CoReg then links the hierarchical clustering of these co-expressed sets with frequency tables of promoter elements. These promoter elements can be drawn from known elements or all possible combinations of nucleotides in an element of various lengths. These sets of promoter elements represent putative cis-acting regulatory elements common to sets of co-expressed genes and can be prioritised for experimental testing. We have used these new tools to analyze the response of transcripts for nuclear genes encoding mitochondrial proteins in Arabidopsis to a range of chemical stresses. ModuleFinder provided a subset of co-expressed gene modules that are more logically related to biological functions than did subsets derived from traditional hierarchical clustering techniques. Importantly ModuleFinder linked responses in transcripts for electron transport chain components, carbon metabolism enzymes and solute transporter proteins. CoReg identified several promoter motifs that helped to explain the patterns of expression observed. CONCLUSION: ModuleFinder identifies sets of genes and treatments that form useful sets for analysis of the mechanisms behind co-expression. CoReg links the clustering tree of expression-based relationships in these sets with frequency tables of promoter elements. These sets of promoter elements represent putative cis-acting regulatory elements for sets of genes, and can then be tested experimentally. We consider these tools, both built on an open source software product to provide valuable, alternative tools for the prioritisation of promoter elements for experimental analysis.
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ItemStat3 regulates microtubules by antagonizing the depolymerization activity of stathmin(ROCKEFELLER UNIV PRESS, 2006-01-16)Stat3 is a member of the signal transducer and activator of transcription family, which is important in cytokine signaling. Gene ablation studies have revealed a requirement for Stat3 in diverse biological processes (Akira, S. 2000. Oncogene. 19: 2607-2611; Levy, D.E., and C.K. Lee. 2002. J. Clin. Invest. 109:1143-1148). Previously, the function of Stat3 had been attributed exclusively to its transcriptional activity in the nucleus. In this study, we reveal an interaction between Stat3 and the microtubule (MT)-destabilizing protein stathmin. Stathmin did not overtly affect ligand-stimulated Stat3 activation. In contrast, the expression of Stat3 is required for the stabilization of MTs and cell migration. We further demonstrate that Stat3-containing cells are resistant to the MT-destabilizing effect of stathmin overexpression. In addition, down-regulation of stathmin protein levels in Stat3-deficient cells partially reversed the MT and migration deficiencies. Recombinant Stat3 was also capable of reversing stathmin inhibition of tubulin polymerization in vitro. Our results indicate that Stat3 modulates the MT network by binding to the COOH-terminal tubulin-interacting domain of stathmin and antagonizing its MT destabilization activity.
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ItemMechanism of filament nucleation and branch stability revealed by the structure of the Arp2/3 complex at actin branch junctions(PUBLIC LIBRARY SCIENCE, 2005-11)Actin branch junctions are conserved cytoskeletal elements critical for the generation of protrusive force during actin polymerization-driven cellular motility. Assembly of actin branch junctions requires the Arp2/3 complex, upon activation, to initiate a new actin (daughter) filament branch from the side of an existing (mother) filament, leading to the formation of a dendritic actin network with the fast growing (barbed) ends facing the direction of movement. Using genetic labeling and electron microscopy, we have determined the structural organization of actin branch junctions assembled in vitro with 1-nm precision. We show here that the activators of the Arp2/3 complex, except cortactin, dissociate after branch formation. The Arp2/3 complex associates with the mother filament through a comprehensive network of interactions, with the long axis of the complex aligned nearly perpendicular to the mother filament. The actin-related proteins, Arp2 and Arp3, are positioned with their barbed ends facing the direction of daughter filament growth. This subunit map brings direct structural insights into the mechanism of assembly and mechanical stability of actin branch junctions.