detection-ligand-activated-g-protein-coupled-receptor-internalization

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

Zhejiang Ocean University

Cathepsin L, a lysosomal endopeptidase, has been noted for its involvement in the innate immune response in invertebrates. Here, the cathepsin L cDNA of the sea cucumber Apostichopus japonicus (AjCatL) is identified from an EST library and then cloned by the rapid amplification of the cDNA ends (RACE) PCR. The full-length cDNA is 1678 bp long containing an open reading frame (ORF) of 1002 bp, an 80 bp 5' UTR and a 599 bp 3' UTR. The cDNA encodes 333 amino acid residues with a predicted molecular mass of 37.07 kDa and a theoretical isoelectric point (pI) of 5.01. The full-length AjCatL contains three active sites of eukaryotic thiol (cysteine) protease at positions 133-144, 278-288 and 295-314. Analysis of the predicted tertiary structure of prepro-CatL (17-333 aa) and mature-CatL (116-333 aa) reveals that the propeptide region (17-115 aa) blocks access to the substrate-binding cleft. Phylogenetic analysis shows that the AjCatL is clustered together with two other CatLs from Strongylocentrotus purpuratus. The enzymatic activity of AjCatL was verified using a substrate hydrolyzing assay with recombinant mAjCatL. Further analysis of real time-PCR demonstrates that the expression of AjCatL mRNA is significantly up-regulated in the coelomocytes in cases of infection with the common bacterial pathogen, Vibrio splendidus. This suggests that the AjCatL is likely to be involved in the immune response.

Cathepsin L of the sea cucumber Apostichopus japonicus-molecular characterization and transcriptional response to Vibrio splendidus infection.

Yohimbine is a novel compound for the treatment of erectile dysfunction derived from natural products, and pharmacokinetic study is important for its further development as a new medicine. In this work, we developed a novel PEEK tube-based solid-phase microextraction (SPME)-HPLC method for analysis of yohimbine in plasma and further for pharmacokinetic study. Poly (AA-EGDMA) was synthesized inside a PEEK tube as the sorbent for microextraction of yohimbine, and parameters that could influence extraction efficiency were systematically investigated. Under optimum conditions, the PEEK tube-based SPME method exhibits excellent enrichment efficiency towards yohimbine. By using berberine as internal standard, an online SPME-HPLC method was developed for analysis of yohimbine in human plasma sample. The method has wide linear range (2-1000 ng/mL) with an R(2) of 0.9962; the limit of detection was determined and was as low as 0.1 ng/mL using UV detection. Finally, a pharmacokinetic study of yohimbine was carried out by the online SPME-HPLC method and the results have been compared with those of reported methods.

PEEK tube-based online solid-phase microextraction-high-performance liquid chromatography for the determination of yohimbine in rat plasma and its application in pharmacokinetics study.

Xiaowei Xiang

National Engineering Research Center of Marine Facilities Aquaculture,
Marine Science College,
National Engineering Research Center of Marine Facilities Aquaculture, Marine Science College

Zhejiang Ocean University

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Xiaowei Xiang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}National Engineering Research Center of Marine Facilities Aquaculture,Marine Science College,National Engineering Research Center of Marine Facilities Aquaculture, Marine Science College

Zhejiang Ocean University

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

National Engineering Research Center of Marine Facilities Aquaculture,
Marine Science College,
National Engineering Research Center of Marine Facilities Aquaculture, Marine Science College