cAMP differentially regulates gamma-globin gene expression in erythroleukemic cells and primary erythroblasts through c-Myb expression

Our previous studies demonstrated roles of cyclic nucleotides in gamma-globin gene expression. We recently found that, upon activation of the cAMP pathway, expression of the gamma-globin gene is inhibited in K562 cells but induced in adult erythroblasts. Here we show that c-Myb, a proto-oncogene product that plays a role in cell growth and differentiation, is involved in the cAMP-mediated differential regulation of gamma-globin gene expression in K562 cells and primary erythroblasts. Our studies found that c-Myb is expressed at a high level in K562 cells compared to primary erythroblasts, and that c-Myb expression is further increased following the treatment with forskolin, an adenylate cyclase activator. The induction of gamma-globin gene expression was also inhibited in K562 cells by raising the levels of c-Myb expression. Importantly, forskolin-induced erythroid differentiation in K562 cells, as determined by the expression of glycophorins and CD71, suggesting that high-level expression of c-Myb may not be sufficient to inhibit the differentiation of erythroid cells. In contrast, c-Myb was not expressed in adult erythroblasts treated with forskolin and primary erythroblasts may lack the c-Myb-mediated inhibitory mechanism for gamma-globin gene expression. Together, these results show that the cAMP pathway blocks gamma-globin gene expression in K562 cells by increasing c-Myb expression and c-Myb plays a role in defining the mode of response of the gamma-globin gene to fetal hemoglobin inducers in erythroid cells.     

Characterization of beta-N-acetylglucosaminidase cleavage by caspase-3 during apoptosis

Beta-O-linked N-acetylglucosamine is a dynamic post-translational modification involved in protein regulation in a manner similar to phosphorylation. Removal of N-acetylglucosamine is regulated by beta-N-acetylglucosaminidase (O-GlcNAcase), which was previously shown to be a substrate of caspase-3 in vitro. Here we show that O-GlcNAcase is cleaved by caspase-3 into two fragments during apoptosis, an N-terminal fragment containing the O-GlcNAcase active site and a C-terminal fragment containing a region with homology to GCN5 histone acetyl-transferases. The caspase-3 cleavage site of O-GlcNAcase, mapped by Edman sequencing, is a noncanonical recognition site that occurs after Asp-413 of the SVVD sequence in human O-GlcNAcase. A point mutation, D413A, abrogates cleavage by caspase-3 both in vitro and in vivo. Finally, we show that O-GlcNAcase activity is not affected by caspase-3 cleavage because the N- and C-terminal O-GlcNAcase fragments remain associated after the cleavage. Furthermore, when co-expressed simultaneously in the same cell, the N-terminal and C-terminal caspase fragments associate to reconstitute O-GlcNAcase enzymatic activity. These studies support the identification of O-GlcNAcase as a caspase-3 substrate with a novel caspase-3 cleavage site and provide insight about O-GlcNAcase regulation during apoptosis.     

A functional genomics approach identifies candidate effectors from the aphid species Myzus persicae (green peach aphid)

Aphids are amongst the most devastating sap-feeding insects of plants. Like most plant parasites, aphids require intimate associations with their host plants to gain access to nutrients. Aphid feeding induces responses such as clogging of phloem sieve elements and callose formation, which are suppressed by unknown molecules, probably proteins, in aphid saliva. Therefore, it is likely that aphids, like plant pathogens, deliver proteins (effectors) inside their hosts to modulate host cell processes, suppress plant defenses, and promote infestation. We exploited publicly available aphid salivary gland expressed sequence tags (ESTs) to apply a functional genomics approach for identification of candidate effectors from Myzus persicae (green peach aphid), based on common features of plant pathogen effectors. A total of 48 effector candidates were identified, cloned, and subjected to transient overexpression in Nicotiana benthamiana to assay for elicitation of a phenotype, suppression of the Pathogen-Associated Molecular Pattern (PAMP)-mediated oxidative burst, and effects on aphid reproductive performance. We identified one candidate effector, Mp10, which specifically induced chlorosis and local cell death in N. benthamiana and conferred avirulence to recombinant Potato virus X (PVX) expressing Mp10, PVX-Mp10, in N. tabacum, indicating that this protein may trigger plant defenses. The ubiquitin-ligase associated protein SGT1 was required for the Mp10-mediated chlorosis response in N. benthamiana. Mp10 also suppressed the oxidative burst induced by flg22, but not by chitin. Aphid fecundity assays revealed that in planta overexpression of Mp10 and Mp42 reduced aphid fecundity, whereas another effector candidate, MpC002, enhanced aphid fecundity. Thus, these results suggest that, although Mp10 suppresses flg22-triggered immunity, it triggers a defense response, resulting in an overall decrease in aphid performance in the fecundity assays. Overall, we identified aphid salivary proteins that share features with plant pathogen effectors and therefore may function as aphid effectors by perturbing host cellular processes.     

Disruption of quorum sensing in seawater abolishes attraction of zoospores of the green alga Ulva to bacterial biofilms

Zoospores of the eukaryotic green seaweed Ulva respond to bacterial N-acylhomoserine lactone (AHL) quorum sensing signal molecules for the selection of surface sites for permanent attachment. In this study we have investigated the production and destruction of AHLs in biofilms of the AHL-producing marine bacterium, Vibrio anguillarum and their stability in seawater. While wild type V. anguillarum NB10 was a strong attractor of zoospores, inactivation of AHL production in this strain by either expressing the recombinant Bacillus lactonase coding gene aiiA, or by mutating the AHL biosynthetic genes, resulted in the abolition of zoospore attraction. In seawater, with a pH of 8.2, the degradation of AHL molecules was temperature-dependent, indicating that the AHLs produced by marine bacterial biofilms have short half-lives. The Ulva zoospores sensed a range of different AHL molecules and in particular more zoospores settled on surfaces releasing AHLs with longer (>six carbons) N-linked acyl chains. However, this finding is likely to be influenced by the differential diffusion rates of AHLs from the experimental surface matrix. Molecules with longer N-acyl chains, such as N-(3-oxodecanoyl)- L-homoserine lactone, diffused more slowly than those with shorter N-acyl chains such as N-(3-hydroxy-hexanoyl)- L-homoserine lactone. Image analysis using GFP-tagged V. anguillarum biofilms revealed that spores settle directly on bacterial cells and in particular on microcolonies which we show are sites of concentrated AHL production.     

Shining Light on Benthic Macroalgae: Mechanisms of Complementarity in Layered Macroalgal Assemblages

Phototrophs underpin most ecosystem processes, but to do this they need sufficient light. This critical resource, however, is compromised along many marine shores by increased loads of sediments and nutrients from degraded inland habitats. Increased attenuation of total irradiance within coastal water columns due to turbidity is known to reduce species'' depth limits and affect the taxonomic structure and architecture of algal-dominated assemblages, but virtually no attention has been paid to the potential for changes in spectral quality of light energy to impact production dynamics. Pioneering studies over 70 years ago showed how different pigmentation of red, green and brown algae affected absorption spectra, action spectra, and photosynthetic efficiency across the PAR (photosynthetically active radiation) spectrum. Little of this, however, has found its way into ecological syntheses of the impacts of optically active contaminants on coastal macroalgal communities. Here we test the ability of macroalgal assemblages composed of multiple functional groups (including representatives from the chlorophyta, rhodophyta and phaeophyta) to use the total light resource, including different light wavelengths and examine the effects of suspended sediments on the penetration and spectral quality of light in coastal waters. We show that assemblages composed of multiple functional groups are better able to use light throughout the PAR spectrum. Macroalgal assemblages with four sub-canopy species were between 50–75% more productive than assemblages with only one or two sub-canopy species. Furthermore, attenuation of the PAR spectrum showed both a loss of quanta and a shift in spectral distribution with depth across coastal waters of different clarity, with consequences to productivity dynamics of diverse layered assemblages. The processes of light complementarity may help provide a mechanistic understanding of how altered turbidity affects macroalgal assemblages in coastal waters, which are increasingly threatened by diminishing light quantity and altered spectral distributions through sedimentation and eutrophication.     

Theoretical study of N<Subscript>4</Subscript>X (X = O,S, Se) systems

A series of N₄X (X = O, S, Se) compounds have been examined with ab initio and density functional theory (DFT) methods. To our knowledge, these compounds, except for the C_2v ring and the C_3v towerlike isomers of N₄O, are first reported here. The ring structures are the most energetically favored for N₄X (X = O and S) systems. For N₄Se, the cagelike structure is the most energetically favored. Several decomposition and isomerization pathways for the N₄X species have been investigated. The dissociation of C_2v ring N₄O and N₄S structures via ring breaking and the barrier height are only 1.1 and −0.2 kcal mol⁻¹ at the CCSD(T)/6-311+G*//MP2/6-311+G* level of theory. The dissociation of the cagelike N₄X species is at a cost of 12.1–16.2 kcal mol⁻¹. As for the towerlike and triangle bipyramidal isomers, their decomposition or isomerization barrier heights are all lower than 10.0 kcal mol⁻¹. Although the C_S cagelike N₄S isomer has a moderate isomerization barrier (18.3–29.1 kcal mol⁻¹), the low dissociation barrier (−1.0 kcal mol⁻¹) indicates that it will disappear when going to the higher CCSD(T) level. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A panel of microsatellite and minisatellite markers for the characterisation of field isolates of Theileria parva

Mini- and microsatellite sequences show high levels of variation and therefore provide excellent tools for both the genotyping and population genetic analysis of parasites. Herein we describe the identification of a panel of 11 polymorphic microsatellites and 49 polymorphic minisatellites of the protozoan haemoparasite Theileria parva. The PCR products were run on high resolution Spreadex gels on which the alleles were identified and sized. The sequences of the mini- and microsatellites were distributed across the four chromosomes with 16 on chromosome 1, 12 on chromosome 2, 14 on chromosome 3 and 18 on chromosome 4. The primers from the 60 sequences were tested against all the Theileria species that co-infect cattle in East and Southern Africa and were found to be specific for T. parva. In order to demonstrate the utility of these markers, we characterised eight tissue culture isolates of T. parva isolated from cattle in widely separated regions of Eastern and Southern Africa (one from Zambia, one from Uganda, two from Zimbabwe, four from Kenya) and one Kenyan tissue culture isolate from Cape buffalo (Syncerus caffer). The numbers of alleles per locus range from three to eight indicating a high level of diversity between these geographically distinct isolates. We also analysed five isolates from cattle on a single farm at Kakuzi in the central highlands of Kenya and identified a range of one to four alleles per locus. Four of the Kakuzi isolates represented distinct multilocus genotypes while two exhibited identical multilocus genotypes. This indicates a high level of diversity in a single population of T. parva. Cluster analysis of multilocus genotypes from the 14 isolates (using a neighbour joining algorithm) revealed that genetic similarity between isolates was not obviously related to their geographical origin.     

Synthesis,Structural and Antioxidant Studies of Some Novel N-Ethyl Phthalimide Esters

A series of N-ethyl phthalimide esters 4(a-n) were synthesized and characterized by spectroscopic studies. Further, the molecular structure of majority of compounds were analysed by single crystal X-ray diffraction studies. The X-ray analysis revealed the importance of substituents on the crystal stability and molecular packing. All the synthesized compounds were tested for in vitro antioxidant activity by DPPH radical scavenging, FRAP and CUPRAC methods. Few of them have shown good antioxidant activity.