Global regulation of alternative splicing during myogenic differentiation

Recent genome-wide analyses have elucidated the extent of alternative splicing (AS) in mammals, often focusing on comparisons of splice isoforms between differentiated tissues. However, regulated splicing changes are likely to be important in biological transitions such as cellular differentiation, or response to environmental stimuli. To assess the extent and significance of AS in myogenesis, we used splicing-sensitive microarray analysis of differentiating C2C12 myoblasts. We identified 95 AS events that undergo robust splicing transitions during C2C12 differentiation. More than half of the splicing transitions are conserved during differentiation of avian myoblasts, suggesting the products and timing of transitions are functionally significant. The majority of splicing transitions during C2C12 differentiation fall into four temporal patterns and were dependent on the myogenic program, suggesting that they are integral components of myogenic differentiation. Computational analyses revealed enrichment of many sequence motifs within the upstream and downstream intronic regions near the alternatively spliced regions corresponding to binding sites of splicing regulators. Western analyses demonstrated that several splicing regulators undergo dynamic changes in nuclear abundance during differentiation. These findings show that within a developmental context, AS is a highly regulated and conserved process, suggesting a major role for AS regulation in myogenic differentiation.     

Genetic analysis of two phosphodiesterases reveals cyclic diguanylate regulation of virulence factors in Dickeya dadantii

Cyclic diguanylate (c‐di‐GMP) is a second messenger implicated in the regulation of various cellular properties in several bacterial species. However, its function in phytopathogenic bacteria is not yet understood. In this study we investigated a panel of GGDEF/EAL domain proteins which have the potential to regulate c‐di‐GMP levels in the phytopathogen Dickeya dadantii 3937. Two proteins, EcpB (contains GGDEF and EAL domains) and EcpC (contains an EAL domain) were shown to regulate multiple cellular behaviours and virulence gene expression. Deletion of ecpB and/or ecpC enhanced biofilm formation but repressed swimming/swarming motility. In addition, the ecpB and ecpC mutants displayed a significant reduction in pectate lyase production, a virulence factor of this bacterium. Gene expression analysis showed that deletion of ecpB and ecpC significantly reduced expression of the type III secretion system (T3SS) and its virulence effector proteins. Expression of the T3SS genes is regulated by HrpL and possibly RpoN, two alternative sigma factors. In vitro biochemical assays showed that EcpC has phosphodiesterase activity to hydrolyse c‐di‐GMP into linear pGpG. Most of the enterobacterial pathogens encode at least one T3SS, a major virulence factor which functions to subvert host defences. The current study broadens our understanding of the interplay between c‐di‐GMP, RpoN and T3SS and the potential role of c‐di‐GMP in T3SS regulation among a wide range of bacterial pathogens.     

Use of polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline for the preparation of a hapten–protein conjugate for antibody development

Polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline (PS-IIDQ), a polymer-supported covalent coupling reagent, was successfully employed for the first time in the bioconjugation of an example hapten (phytanic acid derivative) to a carrier protein (bovine serum albumin (BSA)) within the context of immunogen preparation for antibody development. The ability of the prepared example phytanic acid derivative–BSA conjugate to bind an anti-phytanic acid antibody was confirmed using an enzyme-linked immunosorbent assay (ELISA).     

Lateral asymmetry of paw usage: phenotypic survey of constitutive and experience-conditioned paw-usage behaviours among common strains of the mouse.

Left-right direction of paw usage in the mouse is defined by the right-paw entry (RPE) score, which is the number of reaches with the right paw to retrieve food from a small food tube in a total of 50 right- and left-paw reaches. Two qualitatively different paw-usage behaviours can be identified by the difference in the RPE scores from naive mice in left- or right-biased test chambers and their retest, 1 week later, in the opposite-biased test chamber. In mice with constitutive paw usage, the RPE score may respond to the direction of a biased test chamber, but it returns to the value that is expected for naive mice in the opposite-biased test chamber. In mice with experience-conditioned paw usage, the RPE score responds to the direction of a biased test chamber and does not return to its expected value in the opposite-biased test chamber. In this report, we document the alternate paw usage behaviours in an extended phenotypic survey of different strains that will be useful for its genetic analysis. We also validate an alternate biometrical method to identify constitutive and experience-conditioned paw usage that is based on the mean average RPE score from the biased test and opposite-biased retest of individual mice. This alternate biometrical method demonstrated that, in some strains with experience-conditioned paw usage, there may be asymmetry or an interaction between genotype and the direction of the test sequence. In addition, the strain survey demonstrated that the qualitative difference between constitutive and experience-conditioned paw usage is independent of the well-known quantitative difference in the degree of lateralization of preferred-paw usage.     

NADPH oxidase-dependent reactive oxygen species formation required for root hair growth depends on ROP GTPase

Reactive oxygen species (ROS) production by an NADPH oxidase (NOX) encoded by AtrbohC/RHD2 is required for root hair growth in Arabidopsis thaliana. ROP (RHO of plants) GTPases are also required for normal root hair growth and have been proposed to regulate ROS production in plants. Therefore, the role of ROP GTPase in NOX-dependent ROS formation by root hairs was investigated. Plants overexpressing wild-type ROP2 (ROP2 OX), constitutively active (CA-rop2), or dominant negative (DN-rop2) rop2 mutant proteins were used. Superoxide formation by root hairs was detected by superoxide dismutase-sensitive nitroblue tetrazolium reduction, and ROS production in the root hair differentiation zone was detected by dihydrofluorescein diacetate oxidation. Both probes showed that ROS production was increased in ROP2 OX and CA-rop2 plants, and decreased in DN-rop2 plants, relative to wild-type plants. When CA-rop2 was expressed in the NOX loss-of-function rhd2-1 mutant, ROS formation and root hair growth were impaired, suggesting that RHD2 is required for this ROP2-dependent ROS formation.     

Photooxidation of wetland and riverine dissolved organic matter: altered copper complexation and organic composition

In natural waters, the uptake of transition metals such as copper (Cu) by aquatic biota depends on the activity of the free cupric ion ({Cu²⁺}) rather than on total Cu concentration. Thus, an important ecological function of dissolved organic matter (DOM) in aquatic ecosystems is Cu–DOM complexation, which greatly decreases the {Cu²⁺}. However, Cu bioavailability is greatly modified by source and environmental history of DOM because DOM affinity for Cu varies by orders of magnitude among DOM sources; moreover, DOM is photochemically unstable. During 72-h irradiation experiments at intensities approximating sunlight with DOM from a palustrine wetland and a third-order river, we investigated photooxidative effects on DOM complexation of Cu as well as spectral and chemical changes in DOM that might explain altered Cu complexation. Irradiation decreased Cu complexation by riverine DOM, but unexpectedly increased Cu complexation by wetland DOM, resulting in 150% greater {Cu²⁺} in riverine DOM at the same dissolved organic carbon concentrations. The specific ultraviolet absorption (SUVa) and humic substances tracked photochemical changes in the conditional stability constants of Cu–DOM complexes, suggesting that the aromaticity of DOM influences its affinity for Cu. Carbonyl concentration in ¹³C nuclear magnetic resonance spectra (¹³C-NMR) covaried directly with Cu binding-site densities in DOM. However, no aspect of Cu–DOM complexation consistently covaried with fluorophores (i.e., the fluorescence index) or low molecular weight organic acids. Our results suggest that global increases in UV radiation will affect Cu–DOM complexation and subsequent Cu toxicity depending on light regime as well as DOM source. Handling editor: K. Martens     

Development of photoreceptor-specific promoters and their utility to investigate EIAV lentiviral vector mediated gene transfer to photoreceptors

BACKGROUND: We wanted to investigate the ability of recombinant equine infectious anemia virus (EIAV) vectors to transduce photoreceptor cells by developing a series of photoreceptor-specific promoters that drive strong gene expression in photoreceptor cells. METHODS: Promoter fragments derived from the rhodopsin (RHO), the beta phosphodiesterase (PDE) and the retinitis pigmentosa (RP1) genes were cloned in combination with an enhancer element, derived from the interphotoreceptor retinoid-binding protein gene (IRBP), into luciferase reporter plasmids. An in vitro transient reporter assay was carried out in the human Y-79 retinoblastoma cell line. The optimal promoters from this screen were then cloned into the recombinant EIAV vector for evaluation in vivo following subretinal delivery into mice. RESULTS: All promoters maintained a photoreceptor-specific expression profile in vitro and the gene expression was further enhanced in combination with the IRBP enhancer. The use of IRBP-combined RHO or PDE promoters showed modest but exclusive expression in photoreceptors following subretinal delivery to mice. By contrast an EIAV vector containing the cytomegalovirus (CMV) promoter drove reporter gene expression in both photoreceptors and retinal pigment epithelium. CONCLUSIONS: It may be possible to use recombinant EIAV vectors containing photoreceptor-specific promoters to drive therapeutic gene expression to treat a range of retinal degenerative diseases where the photoreceptor cell is the primary disease target.