Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.     

Attraction of subterranean termites (Isoptera) to carbon dioxide

Subterranean termites, Reticulitermes spp., were attracted to carbon dioxide (CO2) in laboratory and field tests. In behavioral bioassays, Reticulitermes flavipes (Kollar), Reticulitermes tibialis Banks, and Reticulitermes virginicus Banks were attracted to CO2 concentrations between 5 and 50 mmol/mol. In further bioassays, R. tibialis and R. virginicus were attracted to the headspace from polyisocyanurate construction foam that contained 10-12 mmol/mol CO2. In soil bioassays in the laboratory, more termites foraged in chambers containing CO2-generating formulations than in unbaited control chambers. In field tests, stations containing CO2-generating baits attracted R. tibialis away from wooden fence posts at rangeland sites in Colorado. For all of the CO2 formulations tested, termites foraged in significantly more bait stations at treatment fenceposts than in bait stations at the control fenceposts. By the end of the 8-wk study, the number of bait stations located by termites at treatment fenceposts ranged from 40 to 90%. At control fenceposts, termites foraged in only a single station and the one positive station was not located by termites until week 5 of the study. At treatment fenceposts, termites foraged equally in active stations (containing a CO2-generating bait) and passive stations (with no CO2-generating bait), indicating that bait stations may benefit passively from a proximal CO2 source in the soil. CO2 used as an attractant in current baiting systems could improve their effectiveness by allowing earlier exposure of termites to an insecticide.     

Prions adhere to soil minerals and remain infectious

An unidentified environmental reservoir of infectivity contributes to the natural transmission of prion diseases (transmissible spongiform encephalopathies [TSEs]) in sheep, deer, and elk. Prion infectivity may enter soil environments via shedding from diseased animals and decomposition of infected carcasses. Burial of TSE-infected cattle, sheep, and deer as a means of disposal has resulted in unintentional introduction of prions into subsurface environments. We examined the potential for soil to serve as a TSE reservoir by studying the interaction of the disease-associated prion protein (PrP(Sc)) with common soil minerals. In this study, we demonstrated substantial PrP(Sc) adsorption to two clay minerals, quartz, and four whole soil samples. We quantified the PrP(Sc)-binding capacities of each mineral. Furthermore, we observed that PrP(Sc) desorbed from montmorillonite clay was cleaved at an N-terminal site and the interaction between PrP(Sc) and Mte was strong, making desorption of the protein difficult. Despite cleavage and avid binding, PrP(Sc) bound to Mte remained infectious. Results from our study suggest that PrP(Sc) released into soil environments may be preserved in a bioavailable form, perpetuating prion disease epizootics and exposing other species to the infectious agent.     

Conjugation to Nedd8 instigates ubiquitylation and down-regulation of activated receptor tyrosine kinases

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.     

Continuity of interpersonal violence between Nubian communities

Modern communities affiliated with the same culture have been shown to experience comparable levels of interpersonal violence, no matter what their size. It was hypothesized that a similar relationship would exist among ancient rural and urban people, but that accident-related trauma may be more prominent among rural dwellers due to their activity base. Through an analysis of antemortem trauma, this investigation contrasted the injury profile of Nubian adult villagers (N = 55) from the Kerma period (2500-1750 BC) to that of their urban neighbors (N = 223) at Kerma (2050-1500 BC). The injury pattern associated with interpersonal violence (cranial injury, direct-force ulna fractures, and multiple injuries) was similar between the two samples, as hypothesized. The rural group sustained significantly more nonviolence-related injuries that suggested occupational or environmental influences. The more severe cranial injuries observed among urban people are attributed to a preference for more lethal hand-wielded objects that may have accompanied increasing local tensions and incursions into Egypt during the 17th Dynasty.     

Small‐plot studies comparing pheromone and juice baits for mass‐trapping invasive Synanthedon myopaeformis in Canada

Recent introduction of Synanthedon myopaeformis (Borkhausen) (Lepidoptera: Sesiidae) into organic apple‐growing areas of Canada has stimulated research on semiochemical‐based management of this European pest. Replicated, small‐plot (0.16 ha) experiments were conducted to compare sex pheromone, 3Z,13Z‐octadecadienyl acetate (10 mg), Concord grape juice (300 ml), or their combination, as mass‐trapping lures at trap densities equivalent to 12.5, 25, 50, and 100 traps ha^?1. Total numbers of male and female moths removed from test plots increased significantly with trap density in all juice‐based mass‐trapping experiments. In pheromone mass‐trapping experiments, however, total catches of males did not increase significantly as trap densities were increased and catches appeared to plateau with 25–50 traps ha^?1. With pheromone‐based mass‐trapping, significantly fewer males were caught in pheromone‐baited assessment traps at the centre of each mass‐trapping plot than in identical traps in untreated plots. This reduction is indicative of significant trap interference or trap ‘shut‐down’. Increasing the density of juice‐based mass‐trapping had no effect on catches of male or female moths in juice‐baited assessment traps, indicating a short range of attraction and lack of interference between juice traps. Pheromone‐ and juice‐based mass trapping removed similar numbers of males at each trap density tested, respectively, but summed catches of males and females were greatest with juice baits. Combining pheromone and juice into a single mass‐trapping treatment (50 traps ha^?1) did not significantly increase catches of males or females relative to either treatment alone. If a practical bisexual mass‐trapping system is going to be developed for S. myopaeformis, then identification of volatile kairomones in Concord grape juice may be useful.