Label-Free Investigations on the G Protein Dependent Signaling Pathways of Histamine Receptors

G protein activation represents an early key event in the complex GPCR signal transduction process and is usually studied by label-dependent methods targeting specific molecular events. However, the constrained environment of such “invasive” techniques could interfere with biological processes. Although histamine receptors (HRs) represent (evolving) drug targets, their signal transduction is not fully understood. To address this issue, we established a non-invasive dynamic mass redistribution (DMR) assay for the human H_1–4Rs expressed in HEK cells, showing excellent signal-to-background ratios above 100 for histamine (HIS) and higher than 24 for inverse agonists with pEC₅₀ values consistent with literature. Taking advantage of the integrative nature of the DMR assay, the involvement of endogenous Gα_q/11, Gα_s, Gα_12/13 and Gβγ proteins was explored, pursuing a two-pronged approach, namely that of classical pharmacology (G protein modulators) and that of molecular biology (Gα knock-out HEK cells). We showed that signal transduction of hH_1–4Rs occurred mainly, but not exclusively, via their canonical Gα proteins. For example, in addition to Gα_i/o, the Gα_q/11 protein was proven to contribute to the DMR response of hH_3,4Rs. Moreover, the Gα_12/13 was identified to be involved in the hH₂R mediated signaling pathway. These results are considered as a basis for future investigations on the (patho)physiological role and the pharmacological potential of H_1–4Rs.     

A rapid method for enzymatic synthesis and purification of the structured triacylglycerol, 1,3-dilauroyl-2-oleoyl-glycerol

A rapid method for synthesis and purification of the structured triacylglycerol (TAG), 1,3-dilauroyl-2-oleoylglycerol (LaOLa), has been developed. A fraction containing 70% LaOLa was obtained by enzymatic transesterification between triolein and lauric acid using Lipozyme IM, which has 1,3-regioselectivity on the glycerol moiety of TAG. The fraction was passed through a Bond Elut SI Column to remove mono-and diacylglycerols produced during the transesterification. The TAG fraction thus obtained was applied to a reversed-phase column, eluted with acetonitrile/tetrahydrofuran (8:2, vol/vol) to separate different TAG species. The LaOLa fraction obtained by this method was of greater than 99% purity. The concentrations of total fatty acids and fatty acids bound to the sn-2 position of this LaOLa fraction were determined by gas-liquid chromatography after hydrolysis by pancreatic lipase, to confirm the purity of stereospecific isomers in this fraction. The final purity of LaOLa was found to be greater than 95%, which was in good agreement with the result obtained by high-performance liquid chromatography using a Lichrosorb Si60 argentated column.     

Improvement of high temperature stability of Pd coating on Ta by HfN intermediate layer

Hafnium nitride (HfN) was chosen as a material for non-porous intermediate layer to improve the high temperature stability of Pd–Ta composite membranes for hydrogen separation. A layer of dense HfN (70 nm) was prepared between Ta substrate and thin Pd film (300 nm), and the high temperature stability of Pd coating was examined by hydrogen absorption experiments at 573 K after the heat treatments at 873 and 973 K. The HfN layer showed obvious hydrogen permeability, though the permeation rate in HfN appeared to be smaller than that in Pd and Ta. In addition, the degradation in coating effects of Pd at elevated temperatures was substantially retarded by HfN layer. Such improved stability was ascribed to retardation of open porosity development in Pd films and interdiffusion between Pd and Ta. It was concluded that HfN is a potential candidate material for intermediate layer to improve high temperature stability of Pd-group 5 metal composite hydrogen separation membranes.     

Photophysical properties and photostability of novel symmetric polycyclicphenazine-type fluorescent dyes and the dye-doped films

Novel symmetric polycyclicphenazine-type fluorescent dyes have been synthesized and characterized in solution and in polymer films. The dyes exhibited two strong absorption bands at around 365-428 nm and 519-567 nm and an intense fluorescence band at around 560-600 nm (Ф = 0.70-0.87) in 1,4-dioxane. The dye-doped polymer films showed excellent wavelength conversion function that efficiently convert ultraviolet and yellow lights into red light (Ф = 0.61-0.92). Moreover, the photostability of the dye-doped PS, PMMA, and PLA films has been investigated.     

Comparison of L- and D-Amino Acids for Bacterial Imaging in Lung Infection Mouse Model

The effectiveness of L- and D-amino acids for detecting the early stage of infection in bacterial imaging was compared. We evaluated the accumulation of ³H-L-methionine (Met), ³H-D-Met, ³H-L-alanine (Ala), and ³H-D-Ala in E. coli EC-14 and HaCaT cells. Biological distribution was assessed in control and lung-infection-model mice with EC-14 using ³H-L- and D-Met, and ¹⁸F-FDG. A maximum accumulation of ³H-L- and D-Met, and ³H-L- and D-Ala occurred in the growth phase of EC-14 in vitro. The accumulation of ³H-L-Met and L-Ala was greater than that of ³H-D-Met and D-Ala in both EC-14 and HaCaT cells. For all radiotracers, the accumulation was greater in EC-14 than in HaCaT cells at early time points. The accumulation was identified at 5 min after injection in EC-14, whereas the accumulation gradually increased in HaCaT cells over time. There was little difference in biodistribution between ³H-L-and D-Met except in the brain. ³H-L- and D-Met were sensitive for detecting areas of infection after the spread of bacteria throughout the body, whereas ¹⁸F-FDG mainly detected primary infection areas. Therefore, ¹¹C-L- and D-Met, radioisotopes that differ only in terms of ³H labeling, could be superior to ¹⁸F-FDG for detecting bacterial infection in lung-infection-model mice.