156 research outputs found

    Real-time DNA microarray analysis

    Get PDF
    We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e. real-time DNA microarrays, enhances the detection dynamic range of conventional systems by being impervious to probe saturation in the capturing spots, washing artifacts, microarray spot-to-spot variations, and other signal amplitude-affecting non-idealities. We demonstrate in both theory and practice that the time-constant of target capturing in microarrays, similar to all affinity-based biosensors, is inversely proportional to the concentration of the target analyte, which we subsequently use as the fundamental parameter to estimate the concentration of the analytes. Furthermore, to empirically validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays

    A BBPā€“Mud2p heterodimer mediates branchpoint recognition and influences splicing substrate abundance in budding yeast

    Get PDF
    The 3ā€² end of mammalian introns is marked by the branchpoint binding protein, SF1, and the U2AF65-U2AF35 heterodimer bound at an adjacent sequence. Baker's yeast has equivalent proteins, branchpoint binding protein (BBP) (SF1) and Mud2p (U2AF65), but lacks an obvious U2AF35 homolog, leaving open the question of whether another protein substitutes during spliceosome assembly. Gel filtration, affinity selection and mass spectrometry were used to show that rather than a U2AF65/U2AF35-like heterodimer, Mud2p forms a complex with BBP without a third (U2AF35-like) factor. Using mutants of MUD2 and BBP, we show that the BBPā€“Mud2p complex bridges partner-specific Prp39p, Mer1p, Clf1p and Smy2p two-hybrid interactions. In addition to inhibiting Mud2p association, the bbpĪ”56 mutation impairs splicing, enhances pre-mRNA release from the nucleus, and similar to a mud2::KAN knockout, suppresses a lethal sub2::KAN mutation. Unexpectedly, rather than exacerbating bbpĪ”56, the mud2::KAN mutation partially suppresses a pre-mRNA accumulation defect observed with bbpĪ”56. We propose that a BBPā€“Mud2p heterodimer binds as a unit to the branchpoint in vivo and serves as a target for the Sub2p-DExD/H-box ATPase and for other splicing factors during spliceosome assembly. In addition, our results suggest the possibility that the Mud2p may enhance the turnover of pre-mRNA with impaired BBP-branchpoint association

    Murine and Bovine Ī³Ī“ T Cells Enhance Innate Immunity against Brucella abortus Infections

    Get PDF
    Ī³Ī“ T cells have been postulated to act as a first line of defense against infectious agents, particularly intracellular pathogens, representing an important link between the innate and adaptive immune responses. Human Ī³Ī“ T cells expand in the blood of brucellosis patients and are active against Brucella in vitro. However, the role of Ī³Ī“ T cells in vivo during experimental brucellosis has not been studied. Here we report TCRĪ“āˆ’/āˆ’ mice are more susceptible to B. abortus infection than C57BL/6 mice at one week post-infection as measured by splenic colonization and splenomegaly. An increase in TCRĪ³Ī“ cells was observed in the spleens of B. abortus-infected C57BL/6 mice, which peaked at two weeks post-infection and occurred concomitantly with diminished brucellae. Ī³Ī“ T cells were the major source of IL-17 following infection and also produced IFN-Ī³. Depletion of Ī³Ī“ T cells from C57BL/6, IL-17RĪ±āˆ’/āˆ’, and GMCSFāˆ’/āˆ’ mice enhanced susceptibility to B. abortus infection although this susceptibility was unaltered in the mutant mice; however, when Ī³Ī“ T cells were depleted from IFN-Ī³āˆ’/āˆ’ mice, enhanced susceptibility was observed. Neutralization of Ī³Ī“ T cells in the absence of TNF-Ī± did not further impair immunity. In the absence of TNF-Ī± or Ī³Ī“ T cells, B. abortus-infected mice showed enhanced IFN-Ī³, suggesting that they augmented production to compensate for the loss of Ī³Ī“ T cells and/or TNF-Ī±. While the protective role of Ī³Ī“ T cells was TNF-Ī±-dependent, Ī³Ī“ T cells were not the major source of TNF-Ī± and activation of Ī³Ī“ T cells following B. abortus infection was TNF-Ī±-independent. Additionally, bovine TCRĪ³Ī“ cells were found to respond rapidly to B. abortus infection upon co-culture with autologous macrophages and could impair the intramacrophage replication of B. abortus via IFN-Ī³. Collectively, these results demonstrate Ī³Ī“ T cells are important for early protection to B. abortus infections

    The succinic dehydrogenase of seedlings

    No full text
    • ā€¦
    corecore