Phylogenetic Distribution and Evolutionary History of Bacterial DEAD-Box Proteins

DEAD-box proteins are found in all domains of life and participate in almost all cellular processes that involve RNA. The presence of DEAD and Helicase_C conserved domains distinguish these proteins. DEAD-box proteins exhibit RNA-dependent ATPase activity in vitro, and several also show RNA helicase activity. In this study, we analyzed the distribution and architecture of DEAD-box proteins among bacterial genomes to gain insight into the evolutionary pathways that have shaped their history. We identified 1,848 unique DEAD-box proteins from 563 bacterial genomes. Bacterial genomes can possess a single copy DEAD-box gene, or up to 12 copies of the gene, such as in Shewanella. The alignment of 1,208 sequences allowed us to perform a robust analysis of the hallmark motifs of DEAD-box proteins and determine the residues that occur at high frequency, some of which were previously overlooked. Bacterial DEAD-box proteins do not generally contain a conserved C-terminal domain, with the exception of some members that possess a DbpA RNA-binding domain (RBD). Phylogenetic analysis showed a separation of DbpA-RBD-containing and DbpA-RBD-lacking sequences and revealed a group of DEAD-box protein genes that expanded mainly in the Proteobacteria. Analysis of DEAD-box proteins from Firmicutes and γ-Proteobacteria, was used to deduce orthologous relationships of the well-studied DEAD-box proteins from Escherichia coli and Bacillus subtilis. These analyses suggest that DbpA-RBD is an ancestral domain that most likely emerged as a specialized domain of the RNA-dependent ATPases. Moreover, these data revealed numerous events of gene family expansion and reduction following speciation.     

Prevalence to Toxoplasma gondii and Sarcocystis spp. in a reintroduced fisher (Martes pennanti) population in Pennsylvania

Understanding the role of disease in population regulation is important to the conservation of wildlife. We evaluated the prevalence of Toxoplasma gondii exposure and Sarcocystis spp. infection in 46 road-killed and accidentally trapper-killed fisher (Martes pennanti) carcasses collected and stored at -20 C by the Pennsylvania Game Commission from February 2002 to October 2008. Blood samples were assayed for T. gondii antibodies using the modified agglutination test (MAT, 1 : 25) and an indirect immunofluorescent antibody test (IFAT, 1 : 128). For genetic analysis, DNA samples were extracted from thoracic and pelvic limb skeletal muscle from each carcass to test for Sarcocystis spp. using 18s-rRNA PCR primers. Antibodies to T. gondii were found in 100% (38 of 38) of the fishers tested by MAT and in 71% (32 of 45) of the fishers tested by IFAT. PCR analysis revealed that 83% (38 of 46) of the fishers were positive for Sarcocystis spp. Sequence analysis of 7 randomly chosen amplicons revealed the fisher sarcocysts had a 98.3% to 99.1% identity to several avian Sarcocystis spp. sequences in GenBank. Data from our study suggest that a high percentage of fishers in Pennsylvania have been exposed to T. gondii and are infected with Sarcocystis spp.     

Characterization of the oligomeric structure of the Ca(2+)-activated Cl- channel Ano1/TMEM16A

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca²⁺-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl⁻ secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca²⁺, suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.     

Antimicrobial peptides increase tolerance to oxidant stress in Drosophila melanogaster

It is well appreciated that reactive oxygen species (ROS) are deleterious to mammals, including humans, especially when generated in abnormally large quantities from cellular metabolism. Whereas the mechanisms leading to the production of ROS are rather well delineated, the mechanisms underlying tissue susceptibility or tolerance to oxidant stress remain elusive. Through an experimental selection over many generations, we have previously generated Drosophila melanogaster flies that tolerate tremendous oxidant stress and have shown that the family of antimicrobial peptides (AMPs) is over-represented in these tolerant flies. Furthermore, we have also demonstrated that overexpression of even one AMP at a time (e.g. Diptericin) allows wild-type flies to survive much better in hyperoxia. In this study, we used a number of experimental approaches to investigate the potential mechanisms underlying hyperoxia tolerance in flies with AMP overexpression. We demonstrate that flies with Diptericin overexpression resist oxidative stress by increasing antioxidant enzyme activities and preventing an increase in ROS levels after hyperoxia. Depleting the GSH pool using buthionine sulfoximine limits fly survival, thus confirming that enhanced survival observed in these flies is related to improved redox homeostasis. We conclude that 1) AMPs play an important role in tolerance to oxidant stress, 2) overexpression of Diptericin changes the cellular redox balance between oxidant and antioxidant, and 3) this change in redox balance plays an important role in survival in hyperoxia.     

Synthesis of [difluoro-(3-alkenylphenyl)-methyl]-phosphonic acids on non-crosslinked polystyrene and their evaluation as inhibitors of PTP1B

A series of [difluoro-(3-alkenylphenyl)-methyl]-phosphonates were prepared on non-crosslinked polystyrene, a soluble polymer support. After cleavage from the support, the resulting phosphonic acids were examined for inhibition with protein tyrosine phosphatase 1B. Compound 20, bearing an alpha,beta-unsaturated allyl ester moiety, was the most potent of this series of compounds, being a reversible, competitive inhibitor with a K(i) of 8.0+/-1.4 microM.     

Latent inhibition as a function of CS intensity in taste aversion learning

An experiment is reported in which the relationship between the intensity of a preexposed stimulus and latent inhibition was investigated, using the taste aversion learning paradigm in rats. Two concentrations of a saline solution (high, 1%; and low, 0.25%) were used during preexposure and conditioning phases in a factorial design. Two control conditions without preexposure were added, one for each stimulus concentration during conditioning. The known effect of conditioned stimulus (CS) intensity during conditioning was confirmed: the more concentrated the solution used in conditioning, the higher the acquisition rate. A direct relationship was observed between the CS intensity used during preexposure and the latent inhibition effect: the more concentrated the solution during preexposure, the lower the acquisition rate of conditioning. The implications of these results for latent inhibition theories are considered.     

‘After the break’: re-conceptualizing ethnicity,national identity and ‘Malaysian-Chinese’ identities

Focusing on ethnic Chinese as cultural citizens of the nation, this paper examines national identity in the context of generational change. In so doing, it connects to colonialist conceptions of identity the dominant framework of ethnicity that operates in Malaysia. It argues that this framework allows for the nationalist imagining of ‘Malaysian-Chinese’ as ‘outsiders’. In probing the complex conceptual relationship between ethnicity, national identity and cultural citizenship, this article asks: How does ‘ethnicity’ enter into negotiations over the ‘national’ in the cultural realm? What are the notions of cultural difference and national otherness that operate in the negative dualisms by which nation and ethnicity are defined? How are these dualisms tied to notions of authenticity and cultural citizenship? Using the novel The Harmony Silk Factory by Malaysian author Tash Aw to address these questions, this paper argues the need to rethink current policies and narratives of ethnic and national identity in Malaysia.