Characterization of 23S rRNA gene mutation in primary and secondary clarithromycin-resistant Helicobacter pylori strains from East China.

BACKGROUND/AIMS: Clarithromycin is an effective antibiotic for treating Helicobacter pylori; however, the development clarithromycin- resistance by multiple strains prevents the eradication of Helicobacter pylori. We aimed to characterize mutations in the 23S rRNA gene of primary clarithromycin-sensitive, primary clarithromycin-resistant and secondary clarithromycin-resistant Helicobacter pylori strains that were collected in East China and elucidate the mechanisms of clarithromycin resistance. MATERIALS AND METHODS: The disk diffusion test and E-test method were used to determine the clarithromycin susceptibility of clinical Helicobacter pylori strains. The 23S rRNA gene fragments were amplified by polymerase chain reaction from 18 primary clarithromycin- resistant strains, 15 primary sensitive strains and 8 secondary clarithromycin-resistant strains. Polymerase chain reaction-products were sequenced to determine mutations of the 23S rRNA gene. RESULTS: We found an A2143G (8 strains) mutation in primary clarithromycin-resistant strains, an A2143T (5 strains) mutation in secondary clarithromycin-resistant strains; but no mutations were found in position 2143 of sensitive strains. A T2182C mutation in primary clarithromycin-sensitive, primary clarithromycinresistant and secondary clarithromycin-resistant strains was found with a prevalence of 86.7% (13 strains), 72.2% (13 strains) or 87.5% (7 strains), respectively. In addition, we found a G2254T (8 strains) and a G2172T (7 strains) mutation in secondary clarithromycin- resistant strains. These point mutations were absent in primary clarithromycin-resistant and -sensitive strains. CONCLUSION: The gene mutation in position 2143 was associated with resistance to clarithromycin, but the mutation was different between primary and secondary clarithromycin-resistant strains. The T2182C mutation was not associated with clarithromycin resistance. Two new hotspot mutations: G2254T and G2172T, in 23S rRNA were discovered in secondary clarithromycin-resistant strains.

Tetrandrine-induced apoptosis in rat primary hepatocytes is initiated from mitochondria: caspases and endonuclease G (Endo G) pathway.

Tetrandrine, a bisbenylisoquinoline alkaloid isolated from the dried root of Stephenia tetrandra (S Moore), possesses a remarkable pharmacological profile. However, the mechanisms of tetrandrine hepatotoxicity remain to be elucidated. In this study, we first proved apoptosis and mitochondrial dysfunction induced by tetrandrine in Sprague-Dawley rat liver in vivo. By further assuming apoptosis as an important mechanism in tetrandrine-induced hepatotoxicity, we focused on mitochondria-initiated apoptosis in primary hepatocytes isolated from Sprague-Dawley male rats. Tetrandrine treatment led to significant release of cytochrome c and downregulation of Bcl-X(L) accompanied by caspase 3 activation, and ultimately, DNA fragmentation. Caspase 3 activation was markedly inhibited by cyclosporin A (CsA) and Ac-DEVD-CHO. Furthermore, Endo G, a caspase-independent apoptotic protein, was detected for its expression and DNase activity. CsA blocked the release both of Endo G and cytochrome c significantly. Additionally, the generation of reactive oxygen species (ROS) increased in a time-dependent manner corresponding with a fall in intracellular GSH content after 10 microM tetrandrine treatment in 4h. Tetrandrine also induced mitochondrial dysfunction indicated by transition of mitochondrial transmembrane potential and decrease of intracellular ATP level. The findings indicated that the caspase-dependent mitochondrial apoptosis pathway was primarily involved in tetrandrine-induced apoptosis in rat primary hepatocytes. In addition, a caspase-independent pathway indicated by Endo G also contributed to apoptosis caused by tetrandrine. Meanwhile, ROS was proved an important inducer in this apoptosis process.