Protective effect of polygoni cuspidati radix and emodin on Vibrio vulnificus cytotoxicity and infection.

Vibrio vulnificus, a good model organism of bacterial septicemia, causes fatal septicemia manifesting a fulminating course and a high mortality rate within days. In order to identify new natural substances preventing V. vulnificus infection, a plant library was screened for inhibiting cytotoxicity to host cells by using Trypan blue staining and LDH assay. We found that Polygoni Cuspidati Radix potently suppressed the acute death of HeLa and RAW264.7 cells in a dose dependent manner. Further studies revealed that Polygoni Cuspidati Radix inhibited V. vulnificus growth and survival in HI broth and seawater, respectively. We confirmed that Polygoni Cuspidati Radix contained high level of emodin by thin layer chromatography (TLC). Emodin showed direct antibacterial activity against V. vulnificus. In addition, emodin prevented the morphologic damages and acute death of HeLa cells caused from V. vulnificus. The safety of Polygoni Cuspidati Radix and emodin to host cells was confirmed by MTT assay. Polygoni Cuspidati Radix and emodin protected mice from V. vulnificus infection.

A modified method for ICSI in the pig: injection of head membrane-damaged sperm using a 3-4 micro m diameter injection pipette.

BACKGROUND: Conventional ICSI to date was focused only on tail membrane damage to achieve sperm immobilization and disruption of the plasma membrane, even though liberation of soluble sperm factors is achieved by disruption of the sperm head membrane. METHODS: A modified method for ICSI was developed: head membrane-damaged spermatozoa aspirated tail or head first were injected into the ooplasm using a 3-4 micro m diameter injection pipette connected to an open-ended aspiration tube regulated by mouth. The efficiency of this modified ICSI was compared with that of conventional ICSI and IVF. RESULTS: When spermatozoa aspirated tail first were injected, a decondensed sperm head was more frequently observed in the oocyte cytoplasm with the modified ICSI (80.0%) than with conventional ICSI (55.7%) or IVF (63.5%) (P < 0.001). The rates of male pronucleus (MPN) formation in the modified ICSI or IVF were significantly higher (50.7 and 39.7%, respectively) than in conventional ICSI (27.9%) (P < 0.001). The rates of survival, cleavage and embryo development to blastocyst were significantly higher in the modified ICSI (71.7, 60.6 and 17.5%) than in conventional ICSI (48.1, 48.7 and 10.5%) (P < 0.001). No significant differences in MPN formation and embryo development to blastocyst were observed between the tail- and head-first sperm aspiration. CONCLUSION: Our results demonstrated that, in the pig, the procedures of pursuing, capturing and immobilizing a spermatozoon and producing deliberate damage to the tail membrane in conventional ICSI were not required in the modified ICSI. We believe that the present study provides sufficient technical advancement to replace conventional ICSI with the modified ICSI, which is more effective and also avoids unnecessary procedures involved in conventional ICSI.