Enhancer-trap detection of expression patterns corresponding to the polar coordinate system in the imaginal discs of Drosophila melanogaster

We have isolated three classes of enhancertrap lines of Drosophila in which lacZ expression patterns in the imaginal discs are consistent with the idea of a polar (radial and angular) coordinate system of positional information. In the first class (HZ76), a circular pattern was expressed transiently during the mid-third instar larval stage when the radial components of the coordinate are probably generated. The expression patterns of the second class (HZ84) were sector-shaped and circular in the leg disc, suggesting a correlation with both radial and angular coordinate values. The expression patterns found in the third class (PZ63 and PZ22) were circular and appeared to reflect radial positional values. Expression in the latter two classes always began in the presumptive dorsal region of the leg disc and gradually spread to the ventral region. These developmental profiles of expression suggested the existence of a centre that initiates patterned gene expression in the presumptive dorsal region of the leg disc. The PZ22 line showed transient expression during tarsal segmentation, suggesting its involvement in tarsal segment formation. We have cloned the PZ22 gene and partially determined its sequence. The deduced amino acid sequence contained a zinc finger motif found in DNA/RNA binding proteins. By in situ hybridization, we determined that the PZ22 gene was transcribed in the leg disc in a pattern identical to that of the lacZ expression. In addition, it was expressed transiently in the embryonic mesoderm during mesoderm segmentation. The PZ22 gene, therefore, may function both in mesodermal segmentation in the embryo and in tarsal segmentation in the imaginal disc.     

Genetics of a-agglutunin function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae cell adhesion protein a-agglutinin is composed of an anchorage subunit (Aga1p) and an adhesion subunit (Aga2p). Although functional a-agglutinin is expressed only by a cells, previous results indicated that AGA1 RNA is expressed in both a and cells after pheromone induction. Expression of the Aga2p adhesion subunit in a cells allowed a-agglutinability, indicating that a cells express the a-agglutinin anchorage subunit, although no role for Aga1p in cells has been identified. Most of the a-specific agglutination-defective mutants isolated previously were defective in AGA1; a single mutant (La199) was a candidate for an aga2 mutant. Expression of AGA2 under PGK control allowed secretion of active Aga2p from control strains but did not complement the La199 agglutination defect or allow secretion of Aga2p from La 199, suggesting that the La199 mutation might identify a new gene required for a-agglutinin function. However, the La199 agglutination defect showed tight linkage to aga2::URA3 and did not complement aga2::URA3 in a/a diploids. The aga2 gene cloned from La199 was nonfunctional and contained an ochre mutation. The inability of pPGK-AGA2 to express functional Aga2p in La199 was shown to result from an additional mutation(s) that reduces expression of plasmid-borne genes. AGA2 was mapped to the left arm of chromosome VII approximately 28 cM from the centromere.     

The response of Drosophila imaginal disc cell lines to ecdysteroids

Summary We have investigated the action of the moulting hormone 20-hydroxy ecdysone (20-HOE) on our leg and wing imaginal disc cell lines. At the morphological level, cells stop dividing and there is some cell death. The remaining cells elongate and aggregate, often producing long processes which form connections between different aggregates. 20-HOE acts within the first one or two days of a passage, at an optimum concentration of 10 ng/ml, this being about 1/100 of the optimum for ecdysone. One cloned wing cell line, C9, has been found to be relatively insensitive to the action of 20-HOE. We have been able to select for resistance to 20-HOE by growing cells in gradually increasing concentrations of hormone followed by passages in hormone-free medium. This has enabled us to isolate a wing cell line C1.8R from its parent cloned line C1.8+. This shows no response to 20-HOE, and cell growth continues even at hormone concentrations as high as 150 ng/ml. We have measured chitin synthesis by the incorporation of radioactive glucosamine into a cell fraction resistant to extensive alkali hydrolysis. The residue was incubated with chitinase, which resulted in a 50% reduction in labelled product. Treatment with 10 ng/ml of 20-HOE dramatically increased chitin synthesis in line C1.8+, but had no effect in the line C1.8R, selected for resistance to hormone. Correspondence to: M.J. Milner     

Significance of absorption, oxidation, and binding to toxicity of four ecdysone agonists in multi-resistant cotton leafworm

Treatment of last-instar larvae of multi-resistant cotton leafworm Spodoptera littoralis with four dibenzoylhydrazines, methoxyfenozide (RH-2485), tebufenozide (RH-5992), halofenozide (RH-0345), and RH-5849, resulted in premature molting leading to death. Methoxyfenozide was the most toxic followed by tebufenozide, halofenozide, and RH-5849. To explain differences in toxicity, especially between multi-resistant and laboratory strains, absorption in the body tissues and oxidative metabolism were tested with 14C-labeled ecdysone agonist and a Lineweaver-Burk assay, respectively. Then to address different compound potencies in multi-resistant strains, the potency of the four ecdysone agonists was measured based on their ability to mimic the natural insect molting hormone, 20-hydroxyecdysone (20E) by inducing evagination in isolated imaginal wing discs. Using monoclonal antibody 9B9, the presence of ecdysteroid receptors in imaginal discs in vitro was confirmed. In parallel, Scatchard plot analysis with whole imaginal wing discs cultured with different concentrations of 3H-labeled ponasterone A indicated no significant difference in affinity and in number of target sites for binding between multi-resistant and susceptible laboratory strains. The four compounds tested caused the effect as agonists of 20E in vitro, and typically the order of their toxicities (LC50s) corresponded with that for evagination-induction with whole imaginal discs.     

Good adhesion properties of probiotics: a potential risk for bacteremia?

The ability to adhere to human intestinal mucus was tested for lactic acid bacteria of clinical blood culture, human fecal and dairy origin. The blood culture isolates were found to adhere better than the dairy strains. Of the Lactobacillus rhamnosus strains (nine clinical, 10 fecal and three dairy), blood culture isolates adhered better than the fecal strains. Although these results indicate a trend for blood culture isolates to bind to intestinal mucus in higher numbers than strains of dairy and human fecal origin, other factors are also likely to be involved in the etiology of lactobacillemia since some of the clinical Lactobacillus isolates exhibited a relatively low level of adhesion.     

双歧杆菌对EPEC和ETEC粘附的竞争抑制作用

观察双歧杆菌与肠上皮细胞系Lovo 细胞粘附后对肠致病性大肠杆菌(EPEC)及产毒性大肠杆菌(ETEC)粘附的竞争性抑制作用。发现双歧杆菌能完全抑制EPEC与ETEC的粘附,这种作用可能是由于双歧杆菌的占位性保护机制,在空间上阻止了病原菌与Lovo细胞的进一步接近     

The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus

Human intestinal glycoproteins extracted from faeces were used as a model for intestinal mucus to investigate adhesion of pathogenic Escherichia coli and Salmonella strains, and the effect of probiotics on this adhesion. S-fimbriated E. coli expressed relatively high adhesion in the mucus model, but the other tested pathogens adhered less effectively. Probiotic strains Lactobacillus GG and L. rhamnosus LC-705 as well as a L. rhamnosus isolated from human faeces were able to slightly reduce S-fimbria-mediated adhesion. Adhesion of S. typhimurium was significantly inhibited by probiotic L. johnsonii LJ1 and L. casei Shirota. Lactobacillus GG and L. rhamnosus (human isolate) increased the adhesion of S. typhimurium suggesting that the pathogen interacts with the probiotic.     

Larva-adult relationships in an ancestral dipteran: A re-examination of sensillar pathways across the antenna and leg anlagen of Chaoborus crystallinus (DeGeer, 1776; Chaoboridae)

 In one of his classical studies on insect metamorphosis, Weismann compared the imaginal anlagen of the ancestral phantom midge, Chaoborus, with those of advanced brachycerans. We have expanded his findings on the relationships between larval and imaginal organs using electron microscopy and cobalt backfilling of the antenna and leg anlagen and the axonal trajectories of corresponding larval sensilla. We show that both primordia are confluent with the larval antennae and ”leg” sensilla (an ancestral Keilin organ), respectively. These fully developed larval organs represent the distal tips of the imaginal anlagen rather than separate cell clusters. The axons of the larval antenna and leg sensilla project across the corresponding anlagen to their target neuromeres within the central nervous system (CNS). Within the discs, nerves composed of these larval axons, developing afferent fibres and efferences ascending from the CNS are found. Both the structure of the primordia and the axonal trajectories thus relate the situation found in advanced brachycerans with that seen in more ancestral insects. In addition, the larval antennae, legs, wings and even the eyes possess very similar afferent pioneer trajectories supporting the idea that the described pattern is generally used in the ontogeny of sensory systems. Received: 30 June 1998 / Accepted: 27 September 1998     

CD14 and apoptosis

In addition to its role as a mediator of innate pro-inflammatory responses following bacterial lipopolysaccharide (LPS) binding, the 55kDa glycosyl-phosphatidylinositol-linked macrophage plasma membrane glycoprotein CD14 is now also known to play a role in phagocytic clearance of apoptotic cells. Although apoptotic cell-associated ligand(s) for CD14 await definition, initial findings suggest that ligand binding occurs close to, or at the same site as, LPS binding. Significantly, in contrast to LPS clearance and in keeping with the non-phlogistic nature of apoptosis, CD14-dependent engulfment of apoptotic cells fails to elicit pro-inflammatory cytokine release from macrophages. Therefore CD14 may be regarded as an innate immune receptor both for microbial products—after binding which activates inflammatory responses—and for self components, which either fail to induce, or alternatively actively suppress, inflammatory responses. Here we review current knowledge of the structure and functions of CD14, its ligands, its possible modes of signal transduction and its place in the panoply of macrophage molecules implicated in apoptotic-cell clearance.     

Monoclonal antibodies to adhesive cell coat glycoproteins secreted by zoospores of the green alga Enteromorpha

Zoospores of Enteromorpha compressa (L.) Grev. secrete an adhesive cell coat which is involved in their attachment to various substrata. Two monoclonal antibodies (mAbs), designated Ent 1 and Ent 6, were raised against settled zoospores displaying secreted adhesive. Both antibodies labelled specifically the anterior region of the cell containing putative adhesive vesicles. During settlement the antigens recognised by both mAbs were secreted but whereas Ent 6 recognised a fibrillar material released within a few minutes of settlement, Ent 1 recognised components which were associated predominantly with the developing cell wall at later time points. Both mAbs also labelled a Golgi-rich region of settled spores, suggesting that these antigens are also synthesised after settlement. Both mAbs labelled the cell walls of vegetative tissue. Competitive enzyme-linked immunosorbent assay indicated that the two antibodies recognise separate, but overlapping epitopes. In spore settlement assays the Ent 6 immunoglobulin strongly reduced initial adhesion at low concentration whereas the inhibitory effects of Ent 1 occurred at later time points. On analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (SDS-PAGE) both MAbs recognised a major buffer- and SDS-soluble, polydisperse 110-kDa antigen. The 110-kDa component was present in extracts of zoospores and sporulating tissue, but absent, in soluble form, from vegetative tissue. Deglycosylation of zoospore extract with anhydrous HF and peptide N-glycosidase digestion, showed that the major 110-kDa antigen is an N-linked glycan, and that the epitope is borne by the protein component. Time-course experiments showed that the Ent 6 antigen became progressively insoluble after zoospore attachment. Taken together, the data indicate that the two antibodies recognise separate but closely related antigens which have distinctive roles in adhesion and cell wall development. Received: 8 February 1999 / Accepted: 26 July 1999