Stability and optimization of canalicular function in hepatocyte couplets

An enriched preparation of rat hepatocyte couplets was obtaied by collagense perfusion and subsequent elutriation (> 85 per cent couplets and triplets; viability of over 95 per cent). Canalicular secretory activity (the ability to accumulate cholyl-lysyl-fluorescein, CLF) was first apparent after 2 h of culture at 37°C and was present in over 80 per cent of the total population after 5–6 h. This remained almost constant for at least 4 h in both elutriated and directly plated cells. Initial storage of freshly prepared couplets at 4°C for up to 6 h prior to incubation had no adverse effect upon secretory function. Reduction of canalicular secretory activity occurred at a concentration of the hepatotoxic agent menadione (IC₅₀ 17 μM) that was lower than that required to induce mild plasma-membrane blebbing (IC₅₀ 43 μM). This study has optimized and characterized the canalicular secretory effectiveness and stability of an enriched preparation of hepatocyte couplets, and established the feasibility of studies of toxic agents on hepatobiliary function in a heterogeneous population of hepatocytes. In this preparation other biochemical parameters can be assessed, thus complementing previous techniques using individual couplets.     

Inhibition of Aspergillus fumigatus and Its Biofilm by Pseudomonas aeruginosa Is Dependent on the Source,Phenotype and Growth Conditions of the Bacterium

Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents.We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.     

The unpredictability of favourability: condition assessment and protected areas in England

The probability that protected areas will deliver their potential for maintaining or enhancing biodiversity is likely to be maximised if they are appropriately and effectively managed. As a result, governments and conservation agencies are devoting much attention to the management of protected areas. In the U.K., the demand for performance accountability has resulted in Public Service Agreements (PSA) that set out targets for government departments to deliver results in return for investments being made. One such target for England is to ensure that all nationally important wildlife sites are in favourable condition by 2010. Here, we tested the hypothesis, of potential strategic importance, that the ecological condition of these sites is predictable from relationships with a range of physical, environmental and demographic variables. We used binary logistic regression to investigate these relationships, using the results of English Nature’s 1997–2003 condition assessment exercise. Generally, sites in unfavourable condition tend to be larger in area, located at higher elevations, but with higher human population density and are more spatially isolated from units of the same habitat. However, despite the range of different parameters included in our models, the extent to which the condition of any given site could be predicted was low. Our results have implications for the delivery of PSA targets, funding allocation, and the location of new protected areas.     

SOCS2 Balances Metabolic and Restorative Requirements during Liver Regeneration

After significant injury, the liver must maintain homeostasis during the regenerative process. We hypothesized the existence of mechanisms to limit hepatocyte proliferation after injury to maintain metabolic and synthetic function. A screen for candidates revealed suppressor of cytokine signaling 2 (SOCS2), an inhibitor of growth hormone (GH) signaling, was strongly induced after partial hepatectomy. Using genetic deletion and administration of various factors we investigated the role of SOCS2 during liver regeneration. SOCS2 preserves liver function by restraining the first round of hepatocyte proliferation after partial hepatectomy by preventing increases in growth hormone receptor (GHR) via ubiquitination, suppressing GH pathway activity. At later times, SOCS2 enhances hepatocyte proliferation by modulating a decrease in serum insulin-like growth factor 1 (IGF-1) that allows GH release from the pituitary. SOCS2, therefore, plays a dual role in modulating the rate of hepatocyte proliferation. In particular, this is the first demonstration of an endogenous mechanism to limit hepatocyte proliferation after injury.     

A New Model for a Carpool Matching Service

Carpooling is an effective means of reducing traffic. A carpool team shares a vehicle for their commute, which reduces the number of vehicles on the road during rush hour periods. Carpooling is officially sanctioned by most governments, and is supported by the construction of high-occupancy vehicle lanes. A number of carpooling services have been designed in order to match commuters into carpool teams, but it known that the determination of optimal carpool teams is a combinatorially complex problem, and therefore technological solutions are difficult to achieve. In this paper, a model for carpool matching services is proposed, and both optimal and heuristic approaches are tested to find solutions for that model. The results show that different solution approaches are preferred over different ranges of problem instances. Most importantly, it is demonstrated that a new formulation and associated solution procedures can permit the determination of optimal carpool teams and routes. An instantiation of the model is presented (using the street network of Guangzhou city, China) to demonstrate how carpool teams can be determined.     

Characterization of the self association of Avian sarcoma virus integrase by analytical ultracentrifugation.

Retroviral integration protein (IN) has been shown to be both necessary and sufficient for the integration of reverse-transcribed retroviral DNA into the host cell DNA. It has been demonstrated that self-assembly of IN is essential for proper function. Analytical ultracentrifugation was used to determine the stoichiometry and free energy of self-association of a full-length IN in various solvents at 23.3 degrees C. Below 8% glycerol, an association stoichiometry of monomer-dimer-tetramer is observed. At salt concentrations above 500 mM, dimer is the dominant species over a wide range of protein concentrations. However, as physiological salt concentrations are approached, tetramer formation is favored. The addition of glycerol to 500 mM NaCl, 20 mM Tris (pH 8.4), 2 mM beta-mercaptoethanol significantly enhances dimer formation with little effect on tetramer formation. Furthermore, as electrostatic shielding is increased by increasing the ionic strength or decreasing the cation size, dimer formation is strengthened while tetramer formation is weakened. Taken together, the data support a model in which dimer formation includes favorable buried surface interactions which are opposed by charge-charge repulsion, while favorable electrostatic interactions contribute significantly to tetramer formation.