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Building designers rely on predictive rendering techniques to design naturally and artificially lit environments. However, despite decades of work on the correctness of global illumination rendering techniques, our ability to accurately predict light levels in buildings-and to do so in a short time frame as part of an iterative design process-remains limited. In this paper, we present a novel approach to parallelizing construction of an irradiance cache over multiple-bounce paths. Relevant points for irradiance calculation based on one or multiple cameras are located by tracing rays through multiple-bounce paths. Irradiance values are then saved to a cache in reverse bounce order so that the irradiance calculation at each bounce samples from previously calculated values. We show by comparison to high-dynamic range photography of a moderately complex space that our method can predict luminance distribution as accurately as R adiance, the most widely validated tool used today for architectural predictive rendering of daylit spaces, and that it is faster by an order of magnitude. [ABSTRACT FROM AUTHOR]

We have investigated the existence of a precoupled form of the distal C-terminal truncated cannabinoid receptor 1 (CB1-417) and heterotrimeric G proteins in a heterologous insect cell expression system. CB1-417 showed higher production levels than the full-length receptor. The production levels obtained in our expression system were double the values reported in the literature. We also observed that at least the distal C-terminus of the receptor was not involved in receptor dimerization, as was predicted in the literature. Using fluorescence resonance energy transfer, we found that CB1-417 and Gαi1β1γ2 proteins were colocalized in the cells. GTPγS binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the Gαi1 protein in the absence of agonists. A CB1-specific antagonist (SR 141716A) inhibited this constitutive activity of the truncated receptor. We found that the CB1-417/Gαi1β1γ2 complex could be solubilized from Sf9 cell membranes and coimmunoprecipitated. In this study, we have proven that the receptor and G proteins can be coexpressed in higher yields using Sf9 cells, and that the protein complex is stable in detergent solution. Thus, our system can be used to produce sufficient quantities of the protein complex to start structural studies. [ABSTRACT FROM AUTHOR]

Abstract: High-level overexpression of G protein-coupled receptors GPCRs in mammalian cells remains a difficult task inspite of newly developed virus based expression systems. Here, we show that the functional expression level of the recombinant bradykinin receptor (B2R) in mammalian cells can be increased up to sixfold just by the addition of dimethylsulphoxide in the culture medium. Total expression level, cellular localization and binding affinity of the recombinant receptor for its endogenous ligand remains unaltered. The strategy presented here, with recombinant B2R as a case example, is applicable to other GPCRs and provides a generic tool to improve the functional expression level of recombinant GPCRs in mammalian cells. [Copyright &y& Elsevier]

We have optimized the expression level of 20 mammalian G protein-coupled receptors (GPCRs) in the methylotrophic yeast Pichia pastoris. We found that altering expression parameters, including growth temperature, and supplementation of the culture medium with specific GPCR ligands, histidine, and DMSO increased the amount of functional receptor, as assessed by ligand binding, by more than eightfold over standard expression conditions. Unexpectedly, we found that the overall amount of GPCR proteins expressed, in most cases, varied only marginally between standard and optimized expression conditions. Accordingly, the optimized expression conditions resulted in a marked fractional increase in the ratio of ligand binding-competent receptor to total expressed receptor. The results of this study suggest a general approach for increasing yields of functional mammalian GPCRs severalfold over standard expression conditions by using a set of optimized expression condition parameters that we have characterized for the Pichia expression system. Overall, we have more than doubled the number of GPCR targets that can be produced in our laboratories in sufficient amounts for structural studies. [ABSTRACT FROM AUTHOR]