Measurement of immunoglobulin synthesis using the ELISPOT assay

We describe a method for the detection of specific antibody-producing cells from either in vitro or in vivo immunization. These techniques are especially useful for detecting antibodies from developing hybridomas. We have successfully used the system to detect isotype-specific antibodies to a variety of bacterial antigens which were produced by heterohybridomas.     

Comparative Genomic Sequence Analysis of the FXR Gene Family: FMR1, FXR1, and FXR2

Mutations in the X-linked gene FMR1 cause fragile X syndrome, the leading cause of inherited mental retardation. Two autosomal paralogs of FMR1 have been identified, and are known as FXR1 and FXR2. Here we describe and compare the genomic structures of the mouse and human genes FMR1, FXR1, and FXR2. All three genes are very well conserved from mouse to human, with identical exon sizes for all but two FXR2 exons. In addition, the three genes share a conserved gene structure, suggesting they are derived from a common ancestral gene. As a first step towards exploring this hypothesis, we reexamined the Drosophila melanogaster gene Fmr1, and found it to have several of the same intron/exon junctions as the mammalian FXRs. Finally, we noted several regions of mouse/human homology in the noncoding portions of FMR1 and FXR1. Knowledge of the genomic structure and sequence of the FXR family of genes will facilitate further studies into the function of these proteins.     

Corrosion behaviour, metal release and biocompatibility of implant materials coated by TiO2-sol gel chemistry]

Alloys based on titanium or cobalt have been used as implant materials for decades with good success. Because of their natural oxide layer these alloys reveal good corrosion behaviour. In contact with physiological solution metal release takes place, which can cause inflammation. Coatings can improve the corrosion behaviour. In this study Ti6Al4V and Co28Cr6Mo alloys, which are frequently used as implant materials, were tested. Polished discs of these alloys and polished discs, which were coated with TiO2-layers by sol-gel chemistry, were compared regarding their corrosion behaviour and metal ion releasing. The releasing of Al, V, Ti, Co, Cr and Mo was quantified by ICP-MS analysis. The TiO2-coating reduced the release of all ions except of the Al-ion. Both alloys showed a deviating kinetic of ion releasing. In addition, cell response (cell vitality, cell proliferation, endothelial marker CD31 and actin allocation) of osteoblasts and endothelial cells were investigated.     

The positive and negative conservation impacts of sheep grazing and other disturbances on the vascular plant species and vegetation of lowland subhumid Tasmania

Summary An important conservation question for grazed areas of lowland subhumid Tasmania is ‘what effects do different, practical disturbance regimes have on native vegetation?’ An experiment designed to determine the single and interactive effects of fire and sheep grazing was established at four sites with distinct vegetation types. There were significant interactive effects of fire and sheep grazing on vegetation attributes at all sites. An analysis of published and new data indicated that there were several vascular plant species that appeared dependent on sheep grazing for their persistence in the present landscape, while there were others that were intolerant of this disturbance but required other types of disturbance, such as mowing. However, most native species appeared to survive in a wide variety of disturbance regimes short of ploughing and fertilization. The implications of these results are that a variety of disturbance regimes is necessary to maintain biological diversity in this environment, and that the naturalness of the regime is not necessarily relevant to its use for conservation.     

Binding of TPX2 to Aurora A alters substrate and inhibitor interactions

The Aurora kinases are a family of serine/threonine kinases involved in mitosis. The expression of AurA is ubiquitous and cell cycle regulated. It is overexpressed in many tumor types, including breast, colon, and ovarian. TPX2 is a binding partner and activator of AurA. A fragment of TPX2 (residues 1-43) has been shown to be sufficient for binding, kinase activation, and protection from dephosphorylation. We have shown that the addition of TPX2(1-43) increases the catalytic efficiency of AurA. While TPX2 binding has no effect on the turnover number of AurA and does not change the reaction mechanism (characterized here to be a rapid equilibrium random mechanism), it increases the binding affinity of both ATP and a peptide substrate. We have also demonstrated differences in the inhibitor structure-activity relationship (SAR) in the presence or absence of TPX2(1-43). To better understand the differential SAR, we carried out computer modeling studies to gain insight into the effect of TPX2 on the binding interactions between AurA and inhibitors. Our working hypothesis is that TPX2 binding decreases the size and accessibility of a hydrophobic pocket, adjacent to the ATP site, to inhibitors.     

Drosophila APC2 is a cytoskeletally-associated protein that regulates wingless signaling in the embryonic epidermis.

The tumor suppressor adenomatous polyposis coli (APC) negatively regulates Wingless (Wg)/Wnt signal transduction by helping target the Wnt effector beta-catenin or its Drosophila homologue Armadillo (Arm) for destruction. In cultured mammalian cells, APC localizes to the cell cortex near the ends of microtubules. Drosophila APC (dAPC) negatively regulates Arm signaling, but only in a limited set of tissues. We describe a second fly APC, dAPC2, which binds Arm and is expressed in a broad spectrum of tissues. dAPC2's subcellular localization revealed colocalization with actin in many but not all cellular contexts, and also suggested a possible interaction with astral microtubules. For example, dAPC2 has a striking asymmetric distribution in neuroblasts, and dAPC2 colocalizes with assembling actin filaments at the base of developing larval denticles. We identified a dAPC2 mutation, revealing that dAPC2 is a negative regulator of Wg signaling in the embryonic epidermis. This allele acts genetically downstream of wg, and upstream of arm, dTCF, and, surprisingly, dishevelled. We discuss the implications of our results for Wg signaling, and suggest a role for dAPC2 as a mediator of Wg effects on the cytoskeleton. We also speculate on more general roles that APCs may play in cytoskeletal dynamics.     

Improving monitoring tools for spotted wing drosophila,Drosophila suzukii

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) were trapped in the field using colored plastic sphere traps coated with insect Tangle‐trap. Red and black spheres captured significantly more D. suzukii than white spheres. Translucent deli‐cup traps deployed in cherry orchards and baited with yeast, the Alpha Scents lure, or the Scentry lure captured significantly more flies than the Trécé lure and Suzukii bait; all attractants had poor selectivity for D. suzukii. No‐choice evaluations of attractants conducted in field cages corroborated the cherry orchard field study, though translucent deli‐cup traps provisioned with the yeast bait captured significantly more flies than those baited with the Alpha Scents lure. Red sphere traps baited with the Scentry lure captured 3–6× more flies than the deli‐cup trap baited with the same lure, and 3–4× more flies than the deli‐cup trap baited with yeast bait, demonstrating that a trap integrating both visual and olfactory cues is a superior tool for monitoring D. suzukii. Moreover, this simple sticky, dry trap design requires far less labor and maintenance than does a liquid‐based deli‐cup trap.