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.

Article	Year
Cathepsin L of the sea cucumber Apostichopus japonicus-molecular characterization and transcriptional response to Vibrio splendidus infection. Fish & shellfish immunology\| PubMed ID: 26777896	2016
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. Biomedical chromatography : BMC\| PubMed ID: 27739080	2017

Xiaowei Xiang

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