Huwentoxin-I (HWTX-I) is a novel neurotoxin isolated from the venom of Orinithoctonus huwena. Based on its biological activity, HWTX-I could be developed as a pain-killer for clinical purpose. Production of HWTX-I by the bacterium or yeast expression systems resulted in poor yields and the puriWed protein was proved to have lower biological activity than that of native one. So, for the Wrst time, we introduced a new method to express HWTX-I gene in Sf9 cells using baculovirus expression system. Recombinant HWTX-I was recognized by Western blotting and then puriWed by nickel-chelating aYnity chromatography under native conditions. Recombinant HWTX-I showed identical amino acid sequence as native form and exhibited similar eVect on muscular transmission with that of native form. These results indicate that the baculovirus expression system and native puriWcation strategy are viable ways to produce active HWTX-I.

In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primerand a vector primer.However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem-loop structure and could notbescreenedout. This simplemethodproved to be efficient, reliable, inexpensive and time-saving, andmay be suitable for themolecules for which genespecific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosomewalkingandobtained 16positive results from 17 samples.

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.

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.

The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumour and anti-HIV activities. We have found for the first time that TCS stimulated the production of reactive oxygen species (ROS) in JAR cells (a human choriocarcinoma cell line) in a time- and concentrationdependent manner by using the fluorescent probe 2',7'-dichlorofluorescein diacetate with confocal laser scanning microscopy. ESR spectral studies and the inhibition of ROS formation by the superoxide radical anion (O2 −d) scavenger superoxide dismutase, the H3O2 scavenger catalase and the hydroxyl radical (OHd) scavenger mannitol suggested the involvement of O2−d, H2O2 and OHd. TCS-induced ROS formation was shown to be dependent on the presence of both extracellular and intracellular Ca2+; moreover, ROS production paralleled the intracellular Ca2+ elevation induced by TCS, suggesting that ROS production might be a consequence of Ca2+ signalling. TCS-induced activation of caspase-3 was initiated within 2 h; however, TCS-induced production of ROS was initiated within 5min, suggesting that the production of ROS preceded the activation of caspase-3. Simultaneous observation of the nuclear morphological changes via two-photon laser scanning microscopy and ROS production via confocal laser scanning microscopy revealed that ROS is involved in the apoptosis of JAR cells. The involvement of ROS was also confirmed by the inhibition of TCS-induced cell death by the antioxidant Trolox and the ROS scavengers catalase and mannitol. Diethylenetriaminepenta-acetic acid, an inhibitor of metal-facilitated OHd formation, markedly inhibited TCS-induced cell death, suggesting that TCS induced OHd formation via the Fenton reaction. The finding that ROS is involved in the TCS-induced apoptosis of JAR cells might provide new insight into the antitumour and anti-HIV mechanism of TCS.