Trichosanthin (TCS) is a ribosome-inactivating protein (RIP) which can inhibit the growth of human choriocarcinoma (JAR) cells. There are no clear mechanisms to discover the interaction pathway and cytotoxicity of TCS in JAR cells. In this paper, we showed the distribution and transport of ndogenously expressed TCS in JAR cells. Enhanced Green Fluorescence Protein (EGFP), fused with TCS, was applied as a reporter to track the behavior of TCS in JAR cells. Firstly, we investigated the expression stability of EGFP and physiological effects on JAR cells. A stable cell line expressing EGFP was created, which could reproduce and express EGFP even if transplanted into nude mice. Based on the proved stability and feasibility of EGFP in cultured cells and in vivo, the fusion gene of EGFP and TCS was constructed and transfected into JAR cells by liposome. The fluorescence microscopy showed that TCS-EGFP fusion gene was expressed in JAR cells in 24 to 48 hours and the fluorescence spread in cytoplasm mainly and in nucleus partially, which could trace the distribution and transport of TCS-EGFP in JAR cells. Most of fluorescent cells died after 48 hours for the cytotoxicity of expressed TCS-EGFP. These results first reported a stable expression and tracing method by EGFP in JAR cells, and provided theoretical basis to apply TCS in cancer therapy.

Trichomislin, a novel ribosome-inactivating protein, was cloned from the genome of Trichosanthes kirilowii Maxim. The gene was recombined to prokaryotic expression vector and the protein was puriWed by cation-exchange chromatography. The secondary structure of trichomislin was measured by circular-dichroism analysis and the ratios of α-helices and β-sheets were calculated. Trichomislin could inhibit the synthesis of protein in rabbit reticulocyte lysate systems and its reaction me hanism was to inactivate ribosome as an rRNA N-glycosidase. Antitumor analyses indicated trichomislin induced the apoptosis and inhibited the growth of choriocarcinoma cells. Further investigation showed that trichomislin could bind to and enter choriocarcinoma cells, and then increase the caspase-3 activity in a time-dependent manner. At the same time, the concentration of cytochrome c in cytosol increased while that in mitochondria decreased. These results suggested that trichomislin induced apoptosis by releasing cytochrome c from mitochondria which then triggered the caspase family member activation.

A rice PAL (phenylalanine ammonia-lyase) gene sequence (rPAL-P5), which is highly similar to and likely the same as a previously described rice ZB8PAL gene, including the 50-upstream and exon I coding regions of PAL, was isolated using PCR amplification. The expression of several PALs, including rPAL-P5, was strongly induced following inoculation with Pyricularia oryzae or treatment with a P. oryzae elicitor. To identify the promoter region induced by the P. oryzae elicitor, we constructed and subsequently transformed rPAL-P5 promoter deletion series into rice calli using particle bombardment. Results from both elicitor-inducible reporter gene and gel mobility shift assays demonstrated that the sequence -349 to -256 of the rPAL-P5 promoter includes a cis-element involved in the induction of P. oryzae.

Dozens of PCR-based methods are available for chromosome walking from a known sequence to an unknown region. These methods are of three types: inverse PCR, ligation-mediated PCR and randomly primed PCR. However, none of them has been generally applied for this purpose, because they are either difficult or inefficient. Here we describe a simple and efficient PCR strategy