mef2 encodes the only apparent Drosophila homolog of the vertebrate myocyte-specific enhancer factor 2 (MEF2). We show herein that the Drosophila MEF2 protein is expressed throughout the mesoderm following gastrulation. Later in embryogenesis, its expression is maintained in precursors and differentiated cells of the somatic and visceral musculature, as well as the heart. We have characterized genetic deficiencies and EMS-induced point mutations that result in complete loss of MEF2 protein in homozygous mutant embryos. These embryos exhibit a dramatic absence of myosin heavy chain (MHCJ-expressing myoblasts and lack differentiated muscle fibers. Examination of earlier events of muscle development indicates that the specification and early differentiation of somatic muscle precursors are not affected because even-skipped-, nautilus., and/33-tubu/in-expressing myoblasts are present. However, these partially differentiated cells are unable to undergo further differentiation to form muscle fibers in the absence of me[2. The later aspects of differentiation of the visceral mesoderm and the heart are also disrupted in me[2 mutant embryos, although the specification and early development of these tissues appear unaffected. Midgut morphogenesis is disrupted in the mutant embryos, presumably as a consequence of abnormal development of the visceral mesoderm. In the heart, the cardial cells do not express MHC. These results indicate that MEF2 is required for later aspects of differentiation of the three major types of musculature, which include body wall muscles, gut musculature, and the heart, in the Drosophila embryo.

In Drosophila, the amidated neuropeptide pigment dispersing factor (PDF) is expressed by the ventral subset of lateral pacemaker neurons and is required for circadian locomotor rhythms. Residual rhythmicity in pdf mutants likely reflects the activity of other neurotransmitters. We asked whether other neuropeptides contribute to such auxiliary mechanisms. We used the gal4/UAS system to create mosaics for the neuropeptide amidating enzyme PHM; amidation is a highly specific and widespread modification of secretory peptides in Drosophila. Three different gal4 drivers restricted PHM expression to different numbers of peptidergic neurons. These mosaics displayed aberrant locomotor rhythms to degrees that paralleled the apparent complexity of the spatial patterns. Certain PHM mosaics were less rhythmic than pdf mutants and as severe as per mutants. Additional gal4 elements were added to the weakly rhythmic PHM mosaics. Although adding pdf-gal4 provided only partial improvement, adding the widely expressed tim-gal4 largely restored rhythmicity. These results indicate that, in Drosophila, peptide amidation is required for neuropeptide regulation of behavior. They also support the hypothesis that multiple amidated neuropeptides, acting upstream, downstream, or in parallel to PDF, help organize daily locomotor rhythms.

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Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1 mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective assessment of inflammasome activation as well as enable high-throughput screening for inflammasome modulators.

During apoptosis, the activation of a family of cysteine proteases, or caspases, results in proteolytic cleavage of numerous substrates. Antibody probes specific for neoepitopes on protein fragments generated by caspase cleavage provide a means to monitor caspase activity at the level of the individual cell. Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme involved in DNA repair, is a well-known substrate for caspase-3 cleavage during apoptosis. Its cleavage is considered to be a hallmark of apoptosis. Here, we demonstrate that an affinity-purified polyclonal antibody to the p85 fragment of PARP is specific for apoptotic cells. Western blots show that the antibody recognizes the 85-kDa (p85) fragment of PARP but not full-length PARP. We demonstrate a time course of PARP cleavage and DNA fragmentation in situ using the PARP p85 fragment antibody and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in Jurkat cells treated with anti-Fas. Furthermore, our results indicate that the p85 fragment of PARP resulting from caspase cleavage during apoptosis is rapidly localized outside the condensed chromatin but not in the cytoplasm.

In an insect, the tobacco hornworm Manduca sexta, the cerebral neuropeptide prothoracicotropic hormone (PTTH), the primary effector of postembryonic development, exists as two molecular forms. These two PTTH's elicit characteristic in vitro dose responses of activation of prothoracic glands from different developmental stages, an indication that during development the glands change in their sensitivity to the neurohormones. Both PTTH's are active in a specific in situ bioassay. Since they may be released in situ at stage-specific times to evoke distinctly different developmental responses, the PTTH neuroendocrine axis appears to be an effective system for determining the functions of molecular forms of a neurohormone in the regulation of growth and development.

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