Enhanced transformation of tnt by tobacco plants expressing a bacterial nitroreductase

The manufacture, disposal, and detonation of explosives have resulted in the pollution of large tracts of land and groundwater. Historically, 2,4,6-trinitrotoluene (TNT) is the most widely used military explosive and is toxic to biological systems and recalcitrant to degradation. To examine the feasibility of enhancing the ability of plants to detoxify the explosive TNT, we created transgenic tobacco (Nicotiana tabacum) constitutively expressing the nsfI nitroreductase gene from Enterobacter cloacae. The product of TNT reduction by the nitroreductase was found to be 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT). Characterization of the transgenic lines in sterile, aqueous conditions amended with TNT demonstrated that these plants were able to remove all of the TNT from the medium at an initial concentration of 0.5 mM (113 mg L(-1)) TNT. In contrast, growth was suppressed in wild-type plants at 0.1 mM (23 mg L(-1)). Following uptake, transgenic seedlings transformed TNT predominantly to 4-HADNT and its high levels appeared to correlate with enhanced tolerance and transformation of TNT. Transformation products of TNT were subsequently conjugated to plant macromolecules to a greater degree in transgenic tobacco, indicating enhanced detoxification compared to the wild type.     

Nek5 promotes centrosome integrity in interphase and loss of centrosome cohesion in mitosis

Nek5 is a poorly characterized member of the NIMA-related kinase family, other members of which play roles in cell cycle progression and primary cilia function. Here, we show that Nek5, similar to Nek2, localizes to the proximal ends of centrioles. Depletion of Nek5 or overexpression of kinase-inactive Nek5 caused unscheduled separation of centrosomes in interphase, a phenotype also observed upon overexpression of active Nek2. However, separated centrosomes that resulted from Nek5 depletion remained relatively close together, exhibited excess recruitment of the centrosome linker protein rootletin, and had reduced levels of Nek2. In addition, Nek5 depletion led to loss of PCM components, including γ-tubulin, pericentrin, and Cdk5Rap2, with centrosomes exhibiting reduced microtubule nucleation. Upon mitotic entry, Nek5-depleted cells inappropriately retained centrosome linker components and exhibited delayed centrosome separation and defective chromosome segregation. Hence, Nek5 is required for the loss of centrosome linker proteins and enhanced microtubule nucleation that lead to timely centrosome separation and bipolar spindle formation in mitosis.     

Evaluation of gene targeting by homologous recombination in ovine somatic cells

Mouse models for some human genetic diseases are limited in their applications since they do not accurately reproduce the phenotype of the human disease. It has been suggested that larger animals, for example sheep, might produce more useful models, as some aspects of sheep physiology and anatomy are more similar to those of humans. The development of methods to clone animals from somatic cells provides a potential novel route to generate such large animal models following gene targeting. Here, we assess targeting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in ovine somatic cells using homologous recombination (HR) of targeting constructs with extensive (>11 kb) homology. Electroporation of these constructs into ovine fetal and post-natal fibroblasts generated G418-resistant clones, but none analyzed had undergone HR, suggesting that at least for this locus, it is an extremely inefficient process. Karyotyping of targeted ovine fetal fibroblasts showed them to be less chromosomally stable than post-natal fibroblasts, and, moreover, extended culture periods caused them to senesce, adversely affecting their viability for use as nuclear transfer donor cells. These data stress the importance of donor cell choice in somatic cell cloning and suggest that culture time be kept to a minimum prior to nuclear transfer in order to maximize cell viability.     

The effect of accelerometer location on the classification of single-site forearm mechanomyograms

Background  

Recently, pattern recognition methods have been deployed in the classification of multiple activation states from mechanomyogram (MMG) signals for the purpose of controlling switching interfaces. Given the propagative properties of MMG signals, it has been suggested that MMG classification should be robust to changes in sensor placement. Nonetheless, this purported robustness remains speculative to date. This study sought to quantify the change in classification accuracy, if any, when a classifier trained with MMG signals from the muscle belly, is subsequently tested with MMG signals from a nearby location.     

2,8-Dihydroxyadenine lithiasis in a Japanese patient heterozygous at the adenine phosphoribosyltransferase locus.

All reported cases of 2,8-dihydroxyadenine (DHA) lithiasis have been due to functional homozygous deficiency of adenine phosphoribosyltransferase (APRT). Here we describe the first case of DHA lithiasis in a patient who has functional APRT activity in cultured lymphoblasts. The patient is heterozygous for Japanese-type (type II) APRT deficiency as demonstrated by starch-gel electrophoresis and DNA sequence analysis. We also demonstrate the use of starch-gel electrophoresis for differentiation between the type II mutant enzyme and the wild-type enzyme.     

The BTB-zinc Finger Transcription Factor Abrupt Acts as an Epithelial Oncogene in Drosophila melanogaster through Maintaining a Progenitor-like Cell State

The capacity of tumour cells to maintain continual overgrowth potential has been linked to the commandeering of normal self-renewal pathways. Using an epithelial cancer model in Drosophila melanogaster, we carried out an overexpression screen for oncogenes capable of cooperating with the loss of the epithelial apico-basal cell polarity regulator, scribbled (scrib), and identified the cell fate regulator, Abrupt, a BTB-zinc finger protein. Abrupt overexpression alone is insufficient to transform cells, but in cooperation with scrib loss of function, Abrupt promotes the formation of massive tumours in the eye/antennal disc. The steroid hormone receptor coactivator, Taiman (a homologue of SRC3/AIB1), is known to associate with Abrupt, and Taiman overexpression also drives tumour formation in cooperation with the loss of Scrib. Expression arrays and ChIP-Seq indicates that Abrupt overexpression represses a large number of genes, including steroid hormone-response genes and multiple cell fate regulators, thereby maintaining cells within an epithelial progenitor-like state. The progenitor-like state is characterised by the failure to express the conserved Eyes absent/Dachshund regulatory complex in the eye disc, and in the antennal disc by the failure to express cell fate regulators that define the temporal elaboration of the appendage along the proximo-distal axis downstream of Distalless. Loss of scrib promotes cooperation with Abrupt through impaired Hippo signalling, which is required and sufficient for cooperative overgrowth with Abrupt, and JNK (Jun kinase) signalling, which is required for tumour cell migration/invasion but not overgrowth. These results thus identify a novel cooperating oncogene, identify mammalian family members of which are also known oncogenes, and demonstrate that epithelial tumours in Drosophila can be characterised by the maintenance of a progenitor-like state.     

Rapid microsatellite analysis using discontinuous polyacrylamide gel electrophoresis.

A method for screening large numbers of samples for microsatellites using discontinuous, non-denaturing polyacrylamide gels and rapid fluorescent gel staining is described. Disc electrophoresis on slab gels provides high-resolution of PCR products. It is useful for collecting population data once microsatellite loci have been characterized.     

Geotaxis and phototaxis are not determinant factors for white pine weevil (Col., Curculionidae) oviposition location on intact trees and severed treetops

Abstract:  The objective of this investigation was to determine if external factors such as phototaxis and geotaxis determine oviposition site location by reproductively active female white pine weevils, Pissodes strobi (Peck), on intact trees or cut treetops. In prior studies, light and gravity, both external tree factors, were consistently linked with oviposition location. In this study, the external factors that were varied in whole trees or cut treetops, included two weevil populations and two tree species in upright, inverted and horizontal orientations with thick or thin stems, indoor and outdoor locations, warm and cool, wet and dry, and light and dark conditions. The results show that 100% of the oviposition occurred at or near treetops, regardless of external factors. The results are unequivocal; they are without variance. These findings suggest that the reproductively active female weevil's feeding preference is for primary cortex tissue, an internal tree factor. The primary cortex thickness gradient could account for these weevils' capability to locate treetops, where the thickest primary cortex tissue occurs, even when treetops are not at the highest or brightest locations.