Identification of a TRBD zinc finger-interacting protein in Giardia duodenalis and its regulation of telomerase.

BACKGROUND: Giardia duodenalis causes giardiasis, with diarrhea as the primary symptom. The trophozoite proliferation of this zoonotic parasite is mainly affected by telomerase, although the mechanism of telomerase regulation has not been thoroughly analyzed. METHODS: This study was performed to identify the telomerase RNA-binding domain (TRBD)-interacting protein in G. duodenalis and its regulation of telomerase. Interaction between TRBD and interacting proteins was verified via pulldown assays and co-immunoprecipitation (co-IP) techniques, and the subcellular localization of the protein interactions was determined in vivo via split SNAP-tag labeling. The hammerhead ribozyme was designed to deplete the mRNA of TRBD-interacting proteins. RESULTS: Using TRBD as bait, we identified zinc-finger domain (ZFD)-containing proteins and verified it via pulldown and co-IP experiments. Protein-protein interaction occurred in the nuclei of 293T cells and both nuclei of G. duodenalis. The hammerhead ribozyme depleted ZFD mRNA levels, which reduced the reproduction rate of G. duodenalis, telomerase activity and telomere length. CONCLUSIONS: Our findings suggest that ZFD may regulate telomere function in G. duodenalis nuclei.

A sensitive HPLC-MS method for simultaneous determination of thirteen components in rat plasma and its application to pharmacokinetic study of Tanreqing injection.

A rapid, sensitive and selective method based on high-performance liquid chromatography coupled with Q-Exactive mass spectrometry was developed and validated for simultaneous quantification of thirteen components in rat plasma, including chlorogenic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, luteoloside, isochlorogenic acid A, B and C, scutellarin, baicalin, wogonin, baicalein, phillyrin and forsythoside A. After precipitating proteins from the plasma samples with methanol, chromatographic separation of the thirteen components was achieved by using an XBridge™ C18 column (2.1mm×150mm, 5µm) with the mobile phase consisting of methanol and 0.1% formic acid in water at a flow rate of 0.3mL/min. High-resolution MS quantification was adopted with detection on a Q-Exactive mass spectrometer in full-scan mode, and the results were obtained using a mass extraction window of 10ppm at a mass resolution of 70, 000. All the calibration curves exhibited good linearity (r2>0.991) over the measured ranges. The lower limit of quantitation (LLOQ) was in the range of 1.05-8.13ng/mL. The intra- and inter-day precision (RSD) was less than 11.70% and the accuracy (RE) ranged from -5.58% to 12.29%. No significant matrix effect was observed and the extraction recoveries of all the analytes were more than 79.36%. The developed method was applied to a pharmacokinetic study of the thirteen ingredients in rats after intravenous administration of Tanreqing at three doses of 3, 6 and 12mL/kg. The results indicated that 8 of the 13 components, isochlorogenic acid A, B and C, chlorogenic acid, baicalin, wogonin, luteoloside and forsythoside A, had linear pharmacokinetic properties in the tested dosage range.

[Study on the interactions of genisten esterified derivatives with bovine serum albumin].

Under the imitated physiological conditions (pH = 7.4), the interactions of two novel genistein esterified derivatives, genistein 7-acetylferulic acid ester and genistein 7, 4'-di-acetylferulic acid ester (1 and 2), with bovine serum albumin (BSA) were investigated by the fluorescence and UV-Vis spectroscopy. It was observed that both of them can effectively quench the intrinsic fluorescence of BSA. The results suggested that the fluorescence quenching process of BSA at low concentrations of the compounds may be mainly governed by static quenching mechanisms. The binding constants (KA) and the number of binding sites (n) at different temperatures were calculated. From the thermodynamic parameters, it can be judged that the binding of 1 to BSA involved electrostatic interactions, whereas the binding of 2 to BSA involved hydrogen bonds and Van der Waals forces. The binding average distances r between BSA (donor) and the compounds (acceptor) were determined to be 2.63 nm and 2.92 nm respectively based on the Forster theory. Besides, the interactions of BSA with the compounds did not change the conformation of BSA and the binding of compounds to BSA is near tryptophan subunit via synchronous fluorescence spectrometry.