ADP-Glo is a novel bioluminescent, homogeneous assay for monitoring ADP producing biochemical reactions and thus it is an ideal assay for detecting enzyme activity using a wide variety of substrates. It is a universal assay that can be used with protein kinases, lipid kinases, sugar kinases, and many more kinases as well as ATPases. Because of its high sensitivity, it is suitable for monitoring enzyme activities at very early substrate conversions requiring very low amount of enzymes. Furthermore, as the assay is applicable to a broad range of ATP and substrate concentrations, it is optimal for enzymes that require high ATP and substrate concentrations. This is critical since inhibitor potency has to be demonstrated at the cellular level where ATP is present at millimolar concentrations. ADP-Glo is performed in 2 steps upon completion of kinase reaction: a combined termination of kinase reaction and depletion of remaining ATP in the first step, and conversion of generated ADP to ATP and the newly produced ATP to light output using luciferase/luciferin reaction in the second step. The luminescent signal generated is proportional to the ADP concentration produced and is correlated with the kinase activity. Due to its high signal to background and luminescent readout, this assay is less susceptible to generation of false hits and thus it is applicable to not only primary and secondary screening but also kinase profiling.
Current models of autoimmunity suggest that delayed clearance of apoptotic cells leads to the presentation of apoptotic antigens in the context of inflammatory signals, with resultant autoimmunity. These models implicitly assume that, in contrast to early apoptotic cells (that retain membrane integrity), late apoptotic cells (with compromised membranes) act like necrotic cells (which also lack intact membranes), possibly because of the release of proinflammatory intracellular contents. We showed previously that early apoptotic and necrotic cells induce distinct mitogen-activated protein kinase modules in macrophages with which they interact. Exposure to apoptotic cells led to nearly complete inhibition of both basal and macrophage colony-stimulating factor-induced ERK1/2 by macrophages. In contrast, necrotic cells induced ERK1/2. We show here that apoptotic cells also strongly induced both c-Jun N-terminal kinase and p38, whereas necrotic cells had no detectable effect on c-Jun N-terminal kinase and p38. We also compared the signaling events induced in macrophages by exposure to early apoptotic cells, late apoptotic cells, and necrotic cells. The signaling events induced by late apoptotic cells were identical to and just as potent as those induced by early apoptotic cells. Thus, apoptotic cells are functionally equivalent throughout the cell death process, irrespective of membrane integrity. Moreover, the effects of both early and late apoptotic cells on signaling were dominant over those of necrotic cells. These data show that apoptotic cells do not become proinflammatory upon the loss of membrane integrity and are inconsistent with the notion that delayed clearance alone can lead to autoimmunity.Apoptosis is an active energy-dependent process that generally occurs without inflammation or injury to surrounding tissues (1). Apoptotic cells express surface markers that permit their rapid recognition and ingestion by phagocytes (2, 3). Moreover, the cell membrane of cells undergoing apoptosis remains intact until relatively late (1). Thus, the vast majority of cells dying by apoptosis are cleared by phagocytes while their cell membranes are still intact and before they can release their potentially inflammatory intracellular contents.In this view, the noninflammatory behavior of apoptotic cells is essentially passive in that inflammation is avoided by rapid and efficient clearance of apoptotic cells. In fact, apoptotic cells are also actively antiinflammatory (4, 5). For example, the uptake of apoptotic cells actively inhibits the release of proinflammatory mediators such as interleukin 1 and tumor necrosis factor-␣ by macrophages (m) 2 (4 -6). This contrasts with the effect of necrotic cell uptake, which may lead to m activation and the release of proinflammatory cytokines (7). Based on this differential response to apoptotic versus necrotic cells, antigens derived from cells dying by these two distinct processes are thought to have opposite effects on the activation of T cells (8). The proinflammatory effects of...
Recent evidence indicates that phagocytic clearance of apoptotic cells, initially thought to be a silent event, can modulate macrophage (Mφ) function. We show in this work that phagocytic uptake of apoptotic cells or bodies, in the absence of serum or soluble survival factors, inhibits apoptosis and maintains viability of primary cultures of murine peritoneal and bone marrow Mφ with a potency approaching that of serum-supplemented medium. Apoptotic uptake also profoundly inhibits the proliferation of bone marrow Mφ stimulated to proliferate by M-CSF. While inhibition of proliferation is an unusual property for survival factors, the combination of increased survival and decreased proliferation may aid the Mφ in its role as a scavenger during resolution of inflammation. The ability of apoptotic cells to promote survival and inhibit proliferation appears to be the result of simultaneous activation of Akt and inhibition of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1 and ERK2 (ERK1/2). While several activators of the innate immune system, or danger signals, also inhibit apoptosis and proliferation, danger signals and necrotic cells differ from apoptotic cells in that they activate, rather than inhibit, ERK1/2. These signaling differences may underlie the opposing tendencies of apoptotic cells and danger signals in promoting tolerance vs immunity.
The methyltransferase Glo is nonradioactive, antibody-free and homogenous, universal assay to determine enzyme activity of diverse families of methyltransferases. The assay is formatted to meet the requirements of high-throughput screening in drug discovery programs searching for modulators of methyltransferases.
We present the first images of four debris disks observed in scattered light around the young (4-250 Myr old) M dwarfs TWA 7 and TWA 25, the K6 star HD 35650, and the G2 star HD 377. We obtained these images by reprocessing archival Hubble Space Telescope NICMOS coronagraph data with modern post-processing techniques as part of the Archival Legacy Investigation of Circumstellar Environments (ALICE) program. All four disks appear faint and compact compared with other debris disks resolved in scattered light. The disks around TWA 25, HD 35650, and HD 377 appear very inclined, while TWA 7's disk is viewed nearly face-on. The surface brightness of HD 35650's disk is strongly asymmetric. These new detections raise the number of disks resolved in scattered light around M and late-K stars from one (the AU Mic system) to four. This new sample of resolved disks enables comparative studies of heretofore scarce debris disks around low-mass stars relative to solar-type stars.
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