Residual stability of lipase from Candida rugosain hexane, supercritical CO , and supercritical SF

Lipase from Candida rugosa (EC 3.1.1.3) lost only 15% of its activity when held in supercritical CO and about 10% activity in both supercritical SF and hexane even after two days of incubation at up to 60°C and 82 atm.A pressure of 680 atm resulted in up to 15% loss of enzyme activity in supercritical CO and only about 5% loss of activity in supercritical SF 6 even at 410 atm. There was about 60% decrease in enzyme activity even at 1% water content in supercritical CO . Supercritical SF is a better solvent than supercritical CO and hexane.     

Distinguishing positive selection from neutral evolution: boosting the performance of summary statistics

Summary statistics are widely used in population genetics, but they suffer from the drawback that no simple sufficient summary statistic exists, which captures all information required to distinguish different evolutionary hypotheses. Here, we apply boosting, a recent statistical method that combines simple classification rules to maximize their joint predictive performance. We show that our implementation of boosting has a high power to detect selective sweeps. Demographic events, such as bottlenecks, do not result in a large excess of false positives. A comparison to other neutrality tests shows that our boosting implementation performs well compared to other neutrality tests. Furthermore, we evaluated the relative contribution of different summary statistics to the identification of selection and found that for recent sweeps integrated haplotype homozygosity is very informative whereas older sweeps are better detected by Tajima's π. Overall, Watterson's was found to contribute the most information for distinguishing between bottlenecks and selection.     

Identification of the domains required for direct interaction of the helicase-like and polymerase-like RNA replication proteins of brome mosaic virus.

Brome mosaic virus is a positive-strand RNA virus whose RNA replication requires viral protein 1a, which has putative helicase and capping functions, and 2a, which has putative polymerase function. Since domains of related sequence are conserved in a wide range of plus-strand RNA viruses, analysis of 1a and 2a function should have applicability to many other viruses. We have recently demonstrated that 1a and 2a form a complex in vivo and in vitro. Using immune coprecipitation and mutant polypeptides made in reticulocyte lysates, we have now mapped both the 1a and 2a domains necessary for complex formation. The sequences needed to bind 2a map to the carboxy-terminal helicase-like domain of 1a. Truncated polypeptides containing this domain were able to bind to 2a, while several small insertions in the helicase-like domain disrupted binding. The sequence required for binding 1a lies within a 115-residue subset of the 2a N-terminal segment preceding the polymerase-like domain. Truncations or fusion polypeptides containing this segment can bind 1a. We also determined that highly purified 2a protein made in insect cells can form a complex with highly purified 1a helicase-like domain made in Escherichia coli, suggesting that no other factor is required to mediate 1a-2a interaction. Previous genetic analyses of 1a and 2a are consistent with this mapping and show that the newly defined 1a and 2a binding regions are required for RNA synthesis. The locations of these interacting regions are discussed with regard to models of viral replication and the evolution of positive-strand RNA virus genomes.     

A Few Bad Apples: A Model of Disease Influenced Agent Behaviour in a Heterogeneous Contact Environment

For diseases that infect humans or livestock, transmission dynamics are at least partially dependent on human activity and therefore human behaviour. However, the impact of human behaviour on disease transmission is relatively understudied, especially in the context of heterogeneous contact structures such as described by a social network. Here, we use a strategic game, coupled with a simple disease model, to investigate how strategic agent choices impact the spread of disease over a contact network. Using beliefs that are based on disease status and that build up over time, agents choose actions that stochastically determine disease spread on the network. An agent’s disease status is therefore a function of both his own and his neighbours actions. The effect of disease on agents is modelled by a heterogeneous payoff structure. We find that the combination of network shape and distribution of payoffs has a non-trivial impact on disease prevalence, even if the mean payoff remains the same. An important scenario occurs when a small percentage (called noncooperators) have little incentive to avoid disease. For diseases that are easily acquired when taking a risk, then even when good behavior can lead to disease eradication, a small increase in the percentage of noncooperators (less than 5%) can yield a large (up to 25%) increase in prevalence.     

Population dynamics and the evolution of antifungal drug resistance in Candida albicans

Candida albicans is an important human fungal pathogen. Resistance to all major antifungal agents has been observed in clinical isolates of Candida spp. and is a major clinical challenge. The rise and expansion of drug-resistant mutants during exposure to antifungal agents occurs through a process of adaptive evolution, with potentially complex population dynamics. Understanding the population dynamics during the emergence of drug resistance is important for determining the fundamental principles of how fungal pathogens evolve for resistance. While few detailed reports that focus on the population dynamics of C. albicans currently exist, several important features on the population structure and adaptive landscape can be elucidated from existing evolutionary studies in in vivo and in vitro systems.     

Euphol from Euphorbia tirucalli Negatively Modulates TGF-β Responsiveness via TGF-β Receptor Segregation inside Membrane Rafts

Transforming growth factor-β (TGF-β) responsiveness in cultured cells can be modulated by TGF-β partitioning between lipid raft/caveolae- and clathrin-mediated endocytosis pathways. Lipid rafts are plasma membrane microdomains with an important role in cell survival signaling, and cholesterol is necessary for the lipid rafts’ structure and function. Euphol is a euphane-type triterpene alcohol that is structurally similar to cholesterol and has a wide range of pharmacological properties, including anti-inflammatory and anti-cancer effects. In the present study, euphol suppressed TGF-β signaling by inducing TGF-β receptor movement into lipid-raft microdomains and degrading TGF-β receptors.     

Histone deacetylase complexes: functional entities or molecular reservoirs

Using the dextran-binding domain (DBD) of a type of glucosyltransferase (GTF) from Streptococcus sobrinus, we have developed a novel method for purifying recombinant proteins. DBD-tagged green and red fluorescent proteins as well as the parent GTF and DBD moiety were adsorbed well to commercially available cross-linked dextran (such as Sephadex beads and Sephacryl beads), and eluted efficiently with water-soluble dextran. The purity of the eluted proteins after this one-step affinity purification was 90% or better. The results suggest that DBD can be used as a powerful carrier for purification of various recombinant proteins.     

Thapsigargin and EGTA inhibit endothelin-1-induced glucose transport

We have previously demonstrated that ET-1 may enhance glucose transport in 3T3-L1 adipocytes, secondarily to its stimulatory effect on GLUT1 gene expression by a mitogen-activated protein kinase (MAPK)-dependent pathway. In the present study, we further tested the involvement of Ca²⁺ in glucose uptake in response to ET-1. Among a variety of Ca²⁺-related agents tested, EGTA and thapsigargin were found to suppress both the glucose uptake and intracellular Ca²⁺ mobilization induced by ET-1, as determined by Fura-2 analysis. However, a phospholipase C inhibitor, U73122, also eliminated the intracellular calcium mobilization induced by ET-1, but had no effect on ET-1-stimulated glucose uptake. The finding that neither EGTA nor thapsigargin had any influence on ET-1-induced MAPK activation implies that some mechanism downstream of MAPK activation is involved. Further investigation showed that both agents exerted global inhibitory effects on protein and RNA syntheses. Since both thapsigargin and EGTA may deplete endoplasmic reticulum (ER) Ca²⁺ stores, our results suggest that (1) ET-1-induced glucose transport is independent of ET-1's effect on Ca²⁺ mobilization and (2) depletion of ER Ca²⁺ stores per se may interfere with ET-1's effect on GLUT1 expression.