Pharmacological comparison of a recombinant CB1 cannabinoid receptor with its G(alpha 16) fusion product

The pharmacology of G protein-coupled receptors is widely accepted to depend on the G protein subunit to which the agonist-stimulated receptor couples. In order to investigate whether CB(1) agonist-mediated signal transduction via an engineered G(alpha 16) system is different than that of the G(i/o) coupling normally preferred by the CB(1) receptor, we transfected the human recombinant CB(1) receptor (hCB(1)) or a fusion protein comprising the hCB(1) receptor and G(alpha 16) (hCB(1)-G(alpha 16)) into HEK293 cells. From competition binding studies, the rank order of ligand affinities at the hCB(1)-G(alpha 16) fusion protein was found to be similar to that for hCB(1): HU 210 > CP 55,940 > or = SR 141716A > WIN 55212-2 > anandamide > JWH 015. Agonists increased [(35)S]GTP gamma S binding or inhibited forskolin-stimulated cAMP, presumably by coupling to G(i/o), in cells expressing hCB(1) but not hCB(1)-G(alpha 16). However, an analogous rank order of potencies was observed for these agonists in their ability to evoke increases in intracellular calcium concentration in cells expressing hCB(1)-G(alpha 16) but not hCB(1). These data demonstrate that ligand affinities for the hCB(1) receptor are not affected by fusion to the G(alpha 16) subunit. Furthermore, there is essentially no difference in the function of the hCB(1) receptor when coupled to G(i/o) or G (alpha 16).     

The effect of host size on the sex ratio of Syngaster lepidus, a parasitoid of Eucalyptus longhorned borers (Phoracantha spp.)

The solitary larval ectoparasitoid, Syngaster lepidus Brullé, parasitizes the cryptic larvae of two wood-boring beetles, Phoracantha recurva Newman and Phoracantha semipunctata F. The objective of this study was to determine how the female parasitoids allocated the sex of progeny when presented with larval hosts of uniform size classes. Host size was directly correlated with age of the Phoracantha larval hosts. Groups of Phoracantha larvae of a single age class (2-, 3-, 4-, or 5-week-old) were exposed to parasitoids, and sex ratios of the resulting parasitoid progeny from each host age class were determined. A significant relationship was observed among the sizes of P. recurva and P. semipunctata hosts and the sex ratio of emerging parasitoids. Parasitized 2-week-old beetle larvae of both Phoracantha spp. produced only male S. lepidus progeny, whereas older larval hosts produced increasing proportions of female parasitoids (up to 80% females from 5-week-old hosts). Two-week-old Phoracantha larvae of both species produced fewer parasitoids than host larvae 3–5-week-old. The size of parasitoid progeny consistently increased with host larval age (size), and female parasitoids were larger than males across all host size classes. Male S. lepidus developed in approximately 25 days from 2-week-old hosts, and 19–21 days in 3–5-week-old hosts. Female S. lepidus developed in 22–25 days, with developmental time increasing with host size.     

Exploring routes to stabilize a cationic pyridoxamine in an artificial transaminase: site-directed mutagenesis versus synthetic cofactors

Two artificial transaminases were assembled by linking a pyridoxamine derivative within an engineered fatty acid binding protein. The goal of mimicking a native transamination site by stabilizing a cationic pyridoxamine ring system was approached using two different strategies. First, the scaffold of intestinal fatty acid binding protein (IFABP) was tailored by molecular modeling and site-directed mutagenesis to position a carboxylate group close to the pyridine nitrogen of the cofactor. When these IFABP mutants (IFABP-V60C/L38K/E93E and -V60C/E51K/E93E) proved to be unstable, a second approach was explored. By N-methylation of the pyridoxamine, a cationic cofactor was created and tethered to Cys60 of IFABP-V60C/L38K and -V60C/E51K; this latter strategy had the effect of permanently installing a positive charge on the cofactor. These chemogenetic assemblies catalyze the transamination between alpha-ketoglutarate and various amino acids with enantioselectivities of up to 96% ee. The pH profile of the initial rates is bell shaped and similar to native aminotransferases. The k(cat) values and the turnover numbers for these new constructs are the highest achieved to date in our system. This success was only made possible by the unique flexibility of the underlying enzyme design concept employed, which permits full control of both the protein scaffold and the catalytically active group.     

Comparative genome analysis of potential regulatory elements in the ABCG5-ABCG8 gene cluster

The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. To identify potential regulatory elements, 152 kb of the human ABCG5-ABCG8 gene cluster was sequenced and comparative genome analysis was performed. The two genes are oriented in a head-to-head configuration and are separated by a 374-bp intergenic region, which is highly conserved among several species. Using a reporter construct, the intergenic region was found to act as a bidirectional promoter. A conserved GATA site in the intergenic region was shown by site-directed mutagenesis to act as a repressor for the ABCG5 promoter. The intergenic region was also shown to be partially responsive to treatment by LXR agonists. In summary, several potential regulatory elements were found for the ABCG5 and ABCG8 genes, and the intergenic region was found to act as a bidirectional promoter.     

An unusual orientation for Tyr75 in the active site of the aspartic proteinase from Saccharomyces cerevisiae

The structures of the native Saccharomyces cerevisiae proteinase A have been solved by molecular replacement in the monoclinic and trigonal crystal forms and refined at 2.6-2.7A resolution. These structures agree overall with those of other uninhibited aspartic proteinases. However, an unusual orientation for the side chain of Tyr75, a conserved residue on the flexible "flap" that covers the active site and is important for the activity of these enzymes, was found in the trigonal crystals. A similar conformation of Tyr75 occupying the S1 substrate-binding pocket was previously reported only for chymosin (where it was interpreted as representing a "self-inhibited" state of the enzyme), but for no other aspartic proteinases. Since this orientation of Tyr75 has now been seen in the structures of two members of the family of aspartic proteinases, it might indicate that the placement of that residue in the S1 substrate-binding pocket might have some functional significance, analogous to what was seen for self-inhibited structures of serine proteinases.     

A biomechanical analysis of the acute effects of complex training using lower limb exercises

The aim of this study was to investigate the current practice of complex training using a lower body combination of exercises. It was hypothesized that a bout of heavy resistance exercise (HRE), as typically used in complex training, would lead to enhanced performance (in the form of counter-movement [CMJ] and drop [DJ] jump height) and increased electromyographical (EMG) activity in subsequent plyometric exercise, also typical of complex training. Eight strength trained men performed 2 conditions: HRE or control (no-HRE) in a counter-balanced order. Both conditions involved 4 sets of 6-jump trials (3-CMJ/ 3-DJ). The first set of jumps was used as a baseline. For the HRE condition, 5-squats at 85% of 1 repetition maximum (1RM) followed shortly after the first set, while the second, third and fourth sets followed at 3-, 10-, and 20-minutes post-HRE, respectively. The control condition involved the same procedure, but no exercise separated the first 2-sets. There were no significant main effects (p > 0.05) for any CMJ performance variable or EMG activity regardless of muscle or phase of jump. There were no significant (p > 0.05) main effects of heavy resistance exercise on DJ performance variables. Only the biceps femoris during the propulsive phase of the DJ was significantly higher (p < 0.05) following HRE compared to no-HRE. Some trends in the data were evident and the power of the statistical tests was low. It was concluded that no evidence was found to support the experimental hypothesis although the absence of a treatment effect could not be ruled out. From a practical point of view, undertaking a bout of HRE had no adverse effects on subsequent plyometric performance and so some of the advantages of complex training still remain.     

Performance-based selection of likelihood models for phylogeny estimation

Phylogenetic estimation has largely come to rely on explicitly model-based methods. This approach requires that a model be chosen and that that choice be justified. To date, justification has largely been accomplished through use of likelihood-ratio tests (LRTs) to assess the relative fit of a nested series of reversible models. While this approach certainly represents an important advance over arbitrary model selection, the best fit of a series of models may not always provide the most reliable phylogenetic estimates for finite real data sets, where all available models are surely incorrect. Here, we develop a novel approach to model selection, which is based on the Bayesian information criterion, but incorporates relative branch-length error as a performance measure in a decision theory (DT) framework. This DT method includes a penalty for overfitting, is applicable prior to running extensive analyses, and simultaneously compares all models being considered and thus does not rely on a series of pairwise comparisons of models to traverse model space. We evaluate this method by examining four real data sets and by using those data sets to define simulation conditions. In the real data sets, the DT method selects the same or simpler models than conventional LRTs. In order to lend generality to the simulations, codon-based models (with parameters estimated from the real data sets) were used to generate simulated data sets, which are therefore more complex than any of the models we evaluate. On average, the DT method selects models that are simpler than those chosen by conventional LRTs. Nevertheless, these simpler models provide estimates of branch lengths that are more accurate both in terms of relative error and absolute error than those derived using the more complex (yet still wrong) models chosen by conventional LRTs. This method is available in a program called DT-ModSel.     

Pro-inflammatory effects of Burkholderia cepacia on cystic fibrosis respiratory epithelium

Burkholderia cepacia causes pulmonary infection with high mortality in cystic fibrosis (CF) patients which is likely to involve interaction with respiratory epithelium. In this study the pro-inflammatory properties of B. cepacia were examined using a range of respiratory epithelial cell lines. B. cepacia and cell-free culture supernatants were used to stimulate cell lines with (SigmaCFTE29o- and IB3) and without (A549) the CF transmembrane conductance regulator mutation (CFTR), together with corrected cell lines (C38 and S9). Interleukin (IL)-6 and IL-8, but not GM-CSF or IL-1beta, were released from all the cell lines whereas PGE(2) (prostaglandin E(2)) was released from the A549, IB3 and S9 cell lines only. Nuclear factor (NF)-kappaB activation preceded cytokine release and suppression of NF-kappaB activity diminished cytokine release. These studies indicated that B. cepacia secretory products are potent pro-inflammatory agents for respiratory epithelium and suggest functional CFTR is not required for cytokine or prostanoid responses.     

Specifying functional units and reference flows for comparable alternatives

Goal, Scope, and Background  

Despite documentation of product lifetime, performance, and system dependency issues, requirements for specifying functional units and reference flows in LCA have not been developed. The ISO standards simply note that selection between functions is dependent on the goals and scope of the study, that the functional unit must be clearly defined and measurable, and that the reference flows are the amount of product necessary per functional unit. The goal of this work is to suggest and demonstrate the use a set of requirements for specifying the functional unit and reference flows for comparative LCAs.