Impact of synthetic pyrethroid-sheep dip on the indigenous microflora of animal slurries

The chemical constituents of sheep dip in the UK are currently changing from organophosphate-based to synthetic pyrethroid-based insecticides. As a result, changes are also being made to the methods of disposal of these chemicals in the environment, such that pyrethroid sheep dips must now be diluted in animal slurry or water. To date, there is a lack of quantitative information on the impact of the insecticide on the indigenous microflora of animal slurries. This paper investigated the impact of Bayticol (synthetic pyrethroid sheep dip) over a range of concentrations on selected populations of bacteria within animal slurry. It was found that, with increasing pesticide concentration, there was up to a four orders of magnitude increase in the numbers of faecal coliforms and pathogens, such as putative Salmonella spp. These findings have implications for the disposal of sheep dip-amended animal slurries to land from several aspects: (i) the longevity of putative pathogens in the field may require re-evaluation of the time required before the return of grazing livestock to a slurry-amended field; (ii) the potential for the transfer of pathogenic bacteria and faecal coliforms into human and animal foodchains, and (iii) the increased potential for faecal coliforms being washed into streams, rivers and coastal bathing waters.     

Loss of c-Cbl RING finger function results in high-intensity TCR signaling and thymic deletion

Signaling from the T-cell receptor (TCR) in thymocytes is negatively regulated by the RING finger-type ubiquitin ligase c-Cbl. To further investigate this regulation, we generated mice with a loss-of-function mutation in the c-Cbl RING finger domain. These mice exhibit complete thymic deletion by young adulthood, which is not caused by a developmental block, lack of progenitors or peripheral T-cell activation. Rather, this phenotype correlates with greatly increased expression of the CD5 and CD69 activation markers and increased sensitivity to anti-CD3-induced cell death. Thymic loss contrasts the normal fate of the c-Cbl-/- thymus, even though thymocytes from both mutant mice show equivalent enhancement in proximal TCR signaling, Erk activation and calcium mobilization. Remarkably, only the RING finger mutant thymocytes show prominent TCR-directed activation of Akt. We show that the mutant c-Cbl protein itself is essential for activating this pathway by recruiting the p85 regulatory subunit of PI 3-kinase. This study provides a unique model for analyzing high-intensity TCR signals that cause thymocyte deletion and highlights multiple roles of c-Cbl in regulating this process.     

The adapter type protein CMS/CD2AP binds to the proto-oncogenic protein c-Cbl through a tyrosine phosphorylation-regulated Src homology 3 domain interaction

CMS/CD2AP is a cytoplasmic protein critical for the integrity of the kidney glomerular filtration and the T cell function. CMS contains domains and motifs characteristic for protein-protein interactions, and it is involved in the regulation of the actin cytoskeleton. We report here that the individual SH3 domains of CMS bind to phosphotyrosine proteins of approximately 80, 90, and 180 kDa in cell lysates stimulated with epidermal growth factor. The second SH3 domain of CMS bound specifically to a tyrosine-phosphorylated protein of 120 kDa, which we identified as the proto-oncoprotein c-Cbl. The c-Cbl-binding site for CMS mapped to the carboxyl terminus of c-Cbl and is different from the proline-rich region known to bind SH3-containing proteins. CMS binding to c-Cbl was markedly attenuated in a tyrosine phosphorylation-defective c-Cbl mutant indicating that this interaction is dependent on the tyrosine phosphorylation of CMS. It also implies that CMS interacts with c-Cbl in an inducible fashion upon stimulation of a variety of cell-surface receptors. Immunofluorescence analysis revealed that both proteins colocalize at lamellipodia and leading edges of cells, and we propose that the interaction of CMS with c-Cbl offers a mechanism by which c-Cbl associates and regulates the actin cytoskeleton.     

Environmental Variability and Its Impact on the Reproductive Cycle of Antarctic Krill

"Recruitment potential" in Antarctic krill in the Palmer Long-TermEcological Research (LTER) study region west of the AntarcticPeninsula varied significantly over the 7-yr time series betweenJanuary 1993 and January 1999. Timing of ovarian maturation,the percent of the population reproducing, and individual reproductiveoutput (batch volume, embryo diameter) were measured. Indiceshave been developed to quantify the timing and intensity ofreproduction in Antarctic krill. One finding important to estimatesof population fecundity for this long-lived species is thatthe percent of the population reproducing can vary widely, from10 to 98%. Each season was characterized as having delayed,average or advanced ovarian development. In this study we relatethese indices to direct and indirect indicators of spring orannual food availability. The timing of the spring sea ice retreatand the extent of sea ice in the spring (September through November)appear to significantly affect the intensity and timing of reproductionin the population. Intensity of reproduction was highest under"average" conditions, and oöcyte development fastest withconditions of a late retreat and high spring sea ice extent.     

Anabaena apoflavodoxin hydrogen exchange: on the stable exchange core of the alpha/beta(21345) flavodoxin-like family

An important issue in modern protein biophysics is whether structurally homologous proteins share common stability and/or folding features. Flavodoxin is an archetypal alpha/beta protein organized in three layers: a central beta-sheet (strand order 21345) flanked by helices 1 and 5 on one side and helices 2, 3, and 4 on the opposite side. The backbone internal dynamics of the apoflavodoxin from Anabaena is analyzed here by the hydrogen exchange method. The hydrogen exchange rates indicate that 46 amide protons, distributed throughout the structure of apoflavodoxin, exchange relatively slowly at pH 7.0 (k(ex) < 10(-1) min(-1)). According to their distribution in the structure, protein stability is highest on the beta-sheet, helix 4, and on the layer formed by helices 1 and 5. The exchange kinetics of Anabaena apoflavodoxin was compared with those of the apoflavodoxin from Azotobacter, with which it shares a 48% sequence identity, and with Che Y and cutinase, two other alpha/beta (21345) proteins with no significant sequence homology with flavodoxins. Both similarities and differences are observed in the cores of these proteins. It is of interest that a cluster of a few structurally equivalent residues in the central beta-strands and in helix 5 is common to the cores.     

Tissue Hyperplasia and Enhanced T-Cell Signalling via ZAP-70 in c-Cbl-Deficient Mice

The c-Cbl protein is tyrosine phosphorylated and forms complexes with a wide range of signalling partners in response to various growth factors. How c-Cbl interacts with proteins, such as Grb2, phosphatidylinositol 3-kinase, and phosphorylated receptors, is well understood, but its role in these complexes is unclear. Recently, the Caenorhabditis elegans Cbl homolog, Sli-1, was shown to act as a negative regulator of epidermal growth factor receptor signalling. This finding forced a reassessment of the role of Cbl proteins and highlighted the desirability of testing genetically whether c-Cbl acts as a negative regulator of mammalian signalling. Here we investigate the role of c-Cbl in development and homeostasis in mice by targeted disruption of the c-Cbl locus. c-Cbl-deficient mice were viable, fertile, and outwardly normal in appearance. Bone development and remodelling also appeared normal in c-Cbl mutants, despite a previously reported requirement for c-Cbl in osteoclast function. However, consistent with a high level of expression of c-Cbl in the hemopoietic compartment, c-Cbl-deficient mice displayed marked changes in their hemopoietic profiles, including altered T-cell receptor expression, lymphoid hyperplasia, and primary splenic extramedullary hemopoiesis. The mammary fat pads of mutant female mice also showed increased ductal density and branching compared to those of their wild-type littermates, indicating an unanticipated role for c-Cbl in regulating mammary growth. Collectively, the hyperplastic histological changes seen in c-Cbl mutant mice are indicative of a normal role for c-Cbl in negatively regulating signalling events that control cell growth. Consistent with this view, we observed greatly increased intracellular protein tyrosine phosphorylation in thymocytes following CD3ε cross-linking. In particular, phosphorylation of ZAP-70 kinase in thymocytes was uncoupled from a requirement for CD4-mediated Lck activation. This study provides the first biochemical characterization of any organism that is deficient in a member of this unique protein family. Our findings demonstrate critical roles for c-Cbl in hemopoiesis and in controlling cellular proliferation and signalling by the Syk/ZAP-70 family of protein kinases.