The repressor activity of Even-skipped is highly conserved,and is sufficient to activate engrailed and to regulate both the spacing and stability of parasegment boundaries

During segmentation of the Drosophila embryo, even skipped is required to activate engrailed stripes and to organize odd-numbered parasegments. A 16 kb transgene containing the even skipped coding region can rescue normal engrailed expression, as well as all other aspects of segmentation, in even skipped null mutants. To better understand its mechanism of action, we functionally dissected the Even-skipped protein in the context of this transgene. We found that Even-skipped utilizes two repressor domains to carry out its function. Each of these domains can function autonomously in embryos when fused with the Gal4 DNA-binding domain. A chimeric protein consisting only of the Engrailed repressor domain and the Even-skipped homeodomain, but not the homeodomain alone, was able to restore function, indicating that the repression of target genes is sufficient for even skipped function at the blastoderm stage, while the homeodomain is sufficient to recognize those target genes. When Drosophila Even skipped was replaced by its homologs from other species, including a mouse homolog, they could provide substantial function, indicating that these proteins can recognize similar target sites and also provide repressor activity. Using this rescue system, we show that broad, early even skipped stripes are sufficient for activation of both odd- and even-numbered engrailed stripes. Furthermore, these 'unrefined' stripes organize odd-numbered parasegments in a dose-dependent manner, while the refined, late stripes, which coincide cell-for-cell with parasegment boundaries, are required to ensure the stability of the boundaries.     

A complex array of DNA-binding proteins required for pairing-sensitive silencing by a polycomb group response element from the Drosophila engrailed gene

Regulatory DNA from the Drosophila gene engrailed causes silencing of a linked reporter gene (mini-white) in transgenic Drosophila. This silencing is strengthened in flies homozygous for the transgene and has been called "pairing-sensitive silencing." The pairing-sensitive silencing activities of a large fragment (2.6 kb) and a small subfragment (181 bp) were explored. Since pairing-sensitive silencing is often associated with Polycomb group response elements (PREs), we tested the activities of each of these engrailed fragments in a construct designed to detect PRE activity in embryos. Both fragments were found to behave as PREs in a bxd-Ubx-lacZ reporter construct, while the larger fragment showed additional silencing capabilities. Using the mini-white reporter gene, a 139-bp minimal pairing-sensitive element (PSE) was defined. DNA mobility-shift assays using Drosophila nuclear extracts suggested that there are eight protein-binding sites within this 139-bp element. Mutational analysis showed that at least five of these sites are important for pairing-sensitive silencing. One of the required sites is for the Polycomb group protein Pleiohomeotic and another is GAGAG, a sequence bound by the proteins GAGA factor and Pipsqueak. The identity of the other proteins is unknown. These data suggest a surprising degree of complexity in the DNA-binding proteins required for PSE function.     

Synthesis and SAR of heteroaryl-phenyl-substituted pyrazole derivatives as highly selective and potent canine COX-2 inhibitors

The discovery of heteroaryl-phenyl-substituted pyrazole derivatives as canine selective COX-2 inhibitors is described. Structure-activity relationship (SAR) studies of this class of compounds led to the identification of compound 1 which demonstrated a canine whole blood COX-2 inhibitory IC50 of 12 nM and selectivity ratio of COX-1/COX-2 greater than 4000-fold.     

Denitrification activity,wood loss,and N2O emissions over 9 years from a wood chip bioreactor

Loss of nitrate in subsurface drainage water from agricultural fields is an important problem in the Midwestern United States and elsewhere. One possible strategy for reducing nitrate export is the use of denitrification bioreactors. A variety of experimental bioreactor designs have been shown to reduce nitrate losses in drainage water for periods up to several years. This research reports on the denitrification activity of a wood chip-based bioreactor operating in the field for over 9 years. Potential denitrification activity was sustained over the 9-year period, which was consistent with nitrate removal from drainage water in the field. Denitrification potentials ranged from 8.2 to 34 mg N kg^?1 wood during the last 5 years of bioreactor operation. Populations of denitrifying bacteria were greater in the wood chips than in adjacent subsoil. Loss of wood through decomposition reached 75% at the 90–100 cm depth with a wood half-life of 4.6 years. However, wood loss was less than 20% at 155–170 cm depth and the half-life of this wood was 36.6 years. The differential wood loss at these two depths appears to result from sustained anaerobic conditions below the tile drainage line at 120 cm depth. Pore space concentrations of oxygen and methane support this conjecture. Nitrous oxide exported in tile water from the wood chip bioreactor plots was not significantly higher than N₂O exports in tile water from the untreated control plots, and loss of N₂O from tile water exiting the bioreactor accounted for 0.0062 kg N₂O-N kg^?1 NO₃-N.     

Increasing bacterial disease resistance in plants utilizing antibacterial genes from insects

The introduction of genes into plants encoding potent antibacterial proteins, derived from insects, may significantly augment the level of their resistance to bacterial disease. Using modern techniques, genes of choice can be introduced into plant tissue and this tissue can be manipulated to produce viable plants. The potato has been chosen as the model system, not only because of its plasticity of development, which allows for the relatively easy regeneration of whole plants from transformed tissue, but also because the potato ranks among the top four plants in the world in terms of economic importance.     

Postsynaptic expression of tetanus toxin light chain blocks synaptogenesis in Drosophila.

During the development of the nervous system embryonic neurons are incorporated into neural networks that underlie behaviour. For example, during embryogenesis in Drosophila, motor neurons in every body segment are wired into the circuitry that drives the simple peristaltic locomotion of the larva. Very little is known about the way in which the necessary central synapses are formed in such a network or how their properties are controlled. One possibility is that presynaptic and postsynaptic elements form relatively independently of each other. Alternatively, there might be an interaction between presynaptic and postsynaptic neurons that allows for adjustment and plasticity in the embryonic network. Here we have addressed this issue by analysing the role of synaptic transmission in the formation of synaptic inputs onto identified motorneurons as the locomotor circuitry is assembled in the Drosophila embryo. We targeted the expression of tetanus toxin light chain (TeTxLC) to single identified neurons using the GAL4 system. TeTxLC prevents the evoked release of neurotransmitter by enzymatically cleaving the synaptic-vesicle-associated protein neuronal-Synaptobrevin (n-Syb) [1]. Unexpectedly, we found that the cells that expressed TeTxLC, which were themselves incapable of evoked release, showed a dramatic reduction in synaptic input. We detected this reduction both electrophysiologically and ultrastructurally.