Vibrio vulnificus cytolysin induces apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells via caspase-9/3-dependent pathway.

Vibrio vulnificus cytolysin (VVC) is known to be a pore-forming toxin which shows cytotoxicity for mammalian cells in culture and induces apoptosis in endothelial cells. In order to determine whether VVC induces apoptosis in vascular endothelial cells and tumor cells, the cytotoxicity induced by recombinant VVC (rVVC) and its potential mechanism in HUVEC, SGC-7901 and SMMC-7721 cells were investigated. Our study demonstrated that rVVC induced the release of intracellular K(+) from all the target cells, yet lactate dehydrogenase was not released by rVVC. It indicates that osmotic lysis might not contribute to the cytolysin-induced cytotoxicity. The study also demonstrated that rVVC induced apoptosis in HUVEC, SGC-7901 and SMMC-7721 cells in time- and dosage-dependent manners, which was associated with the activation of caspase-9 and -3, but not caspase-8. During the apoptotic process of the target cells, rVVC labeled with FITC was monitored to attach initially to the surface of the cells and entered the cytoplasma subsequently. These findings suggest that VVC may be not only a pore-forming toxin, but also a transmembrane toxin with powerful ability to induce apoptosis in human vascular endothelial cells and tumor cells.

[Study on the bacterial identification using 16S-23S rRNA gene spacer regions]

OBJECTIVE: To examine the use of PCR utilizing 16S-23S rRNA gene spacer regions in the identification of bacteria. METHODS Primers used in PCR were designed by using the target sequences from the genes encoding 16S-23S rRNA spacer regions. PCR was used for the detection of different standard and clinical bacterial strains. RESULTS Characteristic DNA maps were present after using the PCR to identify 27 standard strains from 27 species. The maps could be directly used for classification of the tested bacterial strains or further differentiated by RFLP. The sensitivity of the PCR may be as high as 2.5 CFU/ml. No non-specific amplification products were observed when using DNA from human PBMC funguses or viruses as templates. Thirty-two strains of bacteria isolated from clinical strains showed DNA maps similar to the DNA maps amplified from standard strains. CONCLUSION The PCR detection of bacteria using 16S-23S rRNA gene spacer regions is sensitive, rapid, specific and accurate for identification of bacteria and provides a new rapid method for determining the clinical diagnosis and the etiology of sepsis.