The GAGA transcription factor of Drosophila melanogaster is ubiquitous and plays multiple roles. Characterization of cDNA clones and detection by domain- specific antibodies has revealed that the 70-90 kDa major GAGA species are encoded by two open reading frames producing GAGA factor proteins of 519 amino acids (GAGA-519) and 581 amino acids (GAGA-581), which share a common N-terminal region that is linked to two different glutamine-rich C-termini. Purified recombinant GAGA-519 and GAGA-581 proteins can form homomeric complexes that bind specifically to a single GAGA sequence in vitro. The two GAGA isoforms also function similarly in transient transactivation assays in tissue culture cells and in chromatin remodeling experiments in vitro . Only GAGA-519 protein accumulates during the first 6 h of embryogenesis. Thereafter, both GAGA proteins are present in nearly equal amounts throughout development; in larval salivary gland nuclei they colocalize completely to specific regions along the euchromatic arms of the polytene chromosomes. Coimmunoprecipitation of GAGA-519 and GAGA-581 from crude nuclear extracts and from mixtures of purified recombinant proteins, indicates direct interactions. We suggest that homomeric complexes of GAGA-519 may function during early embryogenesis; both homomeric and heteromeric complexes of GAGA-519 and GAGA-581 may function later.

BackgroundFine particulate matter originating from traffic correlates with increased morbidity and mortality. An important source of traffic particles is brake wear of cars which contributes up to 20% of the total traffic emissions. The aim of this study was to evaluate potential toxicological effects of human epithelial lung cells exposed to freshly generated brake wear particles.ResultsAn exposure box was mounted around a car's braking system. Lung cells cultured at the air-liquid interface were then exposed to particles emitted from two typical braking behaviours („full stop“ and „normal deceleration“). The particle size distribution as well as the brake emission components like metals and carbons was measured on-line, and the particles deposited on grids for transmission electron microscopy were counted. The tight junction arrangement was observed by laser scanning microscopy. Cellular responses were assessed by measurement of lactate dehydrogenase (cytotoxicity), by investigating the production of reactive oxidative species and the release of the pro-inflammatory mediator interleukin-8. The tight junction protein occludin density decreased significantly (p < 0.05) with increasing concentrations of metals on the particles (iron, copper and manganese, which were all strongly correlated with each other). Occludin was also negatively correlated with the intensity of reactive oxidative species. The concentrations of interleukin-8 were significantly correlated with increasing organic carbon concentrations. No correlation was observed between occludin and interleukin-8, nor between reactive oxidative species and interleukin-8.ConclusionThese findings suggest that the metals on brake wear particles damage tight junctions with a mechanism involving oxidative stress. Brake wear particles also increase pro-inflammatory responses. However, this might be due to another mechanism than via oxidative stress.

Oral zolmitriptan exhibits efficacy and good tolerability in the treatment of menstrually associated migraine. Improvement over placebo was observed as early as 30 minutes following treatment.

SummaryIn hepadnaviruses, reverse transcription is primed by the viral reverse transcriptase (RT) lamivudine) 198 of the Rt reaction, was unable to reverse transcribe the template RNA despite the ε signal. The reverse transcriptase activity was tributary to the new translation of the HBV polymerase, since no Rt activity was detected in the presence of both the mRNA and an inhibitor of the initiation of the protein synthesis. As expected, the use of various dideoxynucleotides for the inhibition of the reverse transcriptase activity revealed that the incorporation of the first nucleotides was a T followed by G and A, respectively. The additional use of the nucleoside analogue 2'-3'-dideoxy-3'-thiacytidine triphosphate (3tC; lamivudine) inhibited the Rt reaction by about 40% in this system. The RT activity of the HBV polymerase was also obtained in the rabbit reticulocyte lysate in which the translation reaction was concomitantly performed with the Rt reaction; however the efficiency was lower than that obtained in the translational extracts prepared from eukaryotic cells grown as monolayers.the above results support the conclusion that, in addition to the presence of the very conserved YMDD catalytic motif in the viral polypeptide and the presence of an ε signal on the template RNA, the reverse transcriptase activity of the HBV polymerase is only detected during a reaction in which the translation and the reverse transcriptase reactions are concomitantly performed. The generation of an in vitro assay for the study of the reverse transcriptase activity of the HBV polymerase will undoubtedly allow broad in vitro screening for new antiviral molecules directed against this important protein. Moreover, it might be employed for the ad hoc in vitro screening of antiviral molecules directed against mutants of the HBV polymerase that do not or poorly respond to the treatment with 3tC or other inhibitory molecules. This represents another novel approach to the putative prevention of the development of liver cancer that can thus reduce or replace the use of ducks, ducklings and rabbit reticulocyte lysate. Materials and Methods PlasmidsPlasmid HH3, used for in vitro expression of full-length HBV reverse transcriptase, was constructed by cloning the polymerase is translated in vitro in a first step and thereafter the reverse transcription activity is analysed in a subsequent reaction involving radiolabelled ribonucleotides. To date, all attempts at producing the biologically active HBV polymerase itself in the reticulocyte lysate system have not been fully convincing (Jeong et al., 1996;Kim and Jung, 1999). It was hypothesised that this might be due to the lack of additional protein factors or divalent cations (Jeong et al., 1996;Li and Tyrrell, 1999). Finally, several attempts have also been made to produce the HBV polymerase in insect cells by employing the baculovirus expression system (in which only a very minor percentage of the polymerase is biologically active) (Lanford et al., 1997), in E. coli (Jeong et al., 199...

Background The leading cause of asthma exacerbation is respiratory viral infection. Innate antiviral defense pathways are altered in the asthmatic epithelium, yet involvement of inflammasome signaling in virus-induced asthma exacerbation is not known. Objective To compare influenza-induced activation of inflammasome and innate immune signaling in human bronchial epithelial cells from asthmatics and non-asthmatics and investigate the role of caspase-1 in epithelial cell antiviral defense. Methods Differentiated primary human bronchial epithelial cells from asthmatics and non-asthmatics were infected with influenza A virus. An inflammasome-specific quantitative real-time polymerase chain reaction array was used to compare baseline and influenza-induced gene expression profiles. Cytokine secretion, innate immune gene expression, and viral replication were compared between human bronchial epithelial cells from asthmatics and non-asthmatics. Immunofluorescence microscopy was used to evaluate caspase-1 and PYCARD co-localization. Tracheal epithelial cells from caspase-1 deficient or wildtype mice were infected with influenza and assessed for antiviral gene expression and viral replication. Results Human bronchial epithelial cells from asthmatics had altered influenza-induced expression of inflammasome-related and innate immune signaling components, which correlated with enhanced production of interlukin-1β, interleukin-6, and tumor necrosis factor-α. Specifically, influenza-induced caspase-1 expression was enhanced and localization differed in human bronchial epithelial cells from asthmatics compared to non-asthmatics. Influenza-infected tracheal epithelial cells from caspase-1 deficient mice had reduced expression of antiviral genes and viral replication. Conclusion Caspase-1 plays an important role in the airway epithelial cell response to influenza infection, which is enhanced in asthmatics and may contribute to the enhanced influenza related pathogenesis observed in vivo.