Overexpression of Arabidopsis CBF1 gene in transgenic tobacco alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance.

A known arabidopsis cDNA clone, the CRT/DRE binding factor 1 (CBF1), was isolated and introduced into tobacco plants. It has been reported that CBF1 is one member of CBF gene family related to low temperature and enhancing low temperature tolerance of plants. In the present work, the transcripts could be detected in the transgenic lines. The photochemical efficiency of PSII (F(v)/F(m)) and the photo-oxidizable P700 in the transgenic lines overexpressing CBF1 were higher than that in the wild type plants during the chilling stress under low irradiance. Similarly, the higher NPQ, higher qf, lower Phi(NF), higher activity of SOD, and lower content of MDA were also detected in the transgenic tobacco lines. Additionally, higher expression levels of Nicotiana tabacum copper/zinc superoxide dismutase (Cu/Zn SOD) were also detected in the transgenic lines. These results suggest that CBF1 protein plays an important role in protection of PSII and PSI during the chilling stress under low irradiance.

Knockdown of SMYD3 by RNA interference down-regulates c-Met expression and inhibits cells migration and invasion induced by HGF.

We previously reported that over-expression of SMYD3, a histone H3-K4 specific di- and tri-methyltransferase, plays a key role in cell viability, adhesion, migration and invasion. In this study, we investigated the mechanisms underlying these phenomena and found that knocking down SMYD3 expression in tumor cells significantly reduced the biological function of HGF and inhibited carcinoma cells migration and invasion. Due to the fact that the proto-oncogene c-Met encodes the high-affinity receptor for HGF, and the HGF-c-Met signaling plays a critical role in the tumor genesis, we further identified the partial correlation between SMYD3 and c-Met. The results showed that high expression of c-Met accompanied with over-expression of SMYD3. Silencing SMYD3 expression in tumor cells by specific shRNAs down-regulated c-Met gene transcription, while over-expressing SMYD3 induced c-Met transcription. Moreover, we demonstrated here that two SMYD3 binding sites within the c-Met core promoter region were significant in the transactivation of c-Met. The present findings provide significant insights into the epigenetic regulatory mechanisms of oncogene c-Met expression, and develop the strategies that may inhibit the progression of cancer migration and invasion.

Downregulation of survivin and activation of caspase-3 through the PI3K/Akt pathway in ursolic acid-induced HepG2 cell apoptosis.

Ursolic acid (UA), a naturally occurring pentacyclic triterpene, is a potent in-vitro anticancer agent, acting through control of growth, apoptosis and differentiation. As the mechanism of its proapoptotic effects on human hepatocellular carcinoma cells has not been extensively studied, we performed an in depth evaluation of the effects of UA on apoptosis in human HepG2 cells. UA was found to inhibit the proliferation of HepG2 cells in a concentration and time-dependent manner. After treatment, cells showed evidence of activation of apoptosis, including the presence of apoptotic bodies and DNA fragmentation. UA-induced apoptosis was accompanied by a significant decrease in bcl-2 and survivin expression, with the corresponding ratio of bax/bcl-2 increased. The treatment with UA also increased the protein level and enzymatic activity of caspase-3. Z-DEVD-fmk, a specific caspase-3 inhibitor, significantly inhibited both the cytotoxic effect and the DNA fragmentation induced by UA, demonstrating the requirement for caspase-3 activity in UA-induced apoptosis. Inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway was also involved, as inhibition of PI3K by LY294002 significantly increased UA-induced apoptosis. Kinetic experiments indicated that UA downregulated PI3K/p85 subunit (PI3K/p85) and phospho-Akt, before downregulating survivin. The further results also confirmed that LY294002 not only downregulated survivin alone, but considerably enhanced the repression of survivin combined with UA. UA therefore seemed to downregulate the expression of survivin by blocking PI3K/Akt. Taken together, the data suggest that the proapoptotic effect of UA on HepG2 cells is mediated by activation of caspase-3, and is highly correlated with inactivation of PI3K/Akt/survivin pathway.

[Functional expression of an omega-3 fatty acid desaturase gene from Glycine max in Saccharomyces cerevisiae].

Alpha-linolenic acid(ALA, C18:3delta9,12,15 ) is an essential fatty acid which has many sanitary functions to human. However, its contents in diets are often not enough. In plants, omega-3 fatty acid desaturases(FAD) catalyze linoleic acid(LA, C18:2delta9,12) into ALA. The seed oil of Glycine max contains high level of ALA. To investigate the functions of Glycine max omega-3FAD, the cDNA of GmFAD3 C was amplified by RT-PCR from immature seeds, then cloned into the shuttle expression vector p416 to generate the recombinant vector p4GFAD3C. The resulting vector was transformed into Saccharomyces cerevisiae K601 throuth LiAc method. The positive clones were screened on the CM(Ura-) medium and identified by PCR, and then cultured in CM (Ura-) liquid medium with exogenous LA in 20 degrees C for three days. The intracellular fatty acid composition of the engineering strain Kp416 and Kp4GFAD3C was analyzed by gas chromatography (GC). A novel peak in strain Kp4GFAD3C was detected,which was not detectable in control, Comparison of the retention times of the newly yielded peak with that of authentic standard indicated that the fatty acid is ALA. The content of ALA reached to 3.1% of the total fatty acid in recombinant strain, the content of LA correspondingly decreased from 22% to 16.2% by contrast. It was suggested that the protein encoded by GmFAD3 C can specifically catalyze 18 carbon PUFA substrate of LA into ALA by taking off hydrogen atoms at delta15 location. In this study, we expressed a Glycine max omega-3 fatty acid desaturase gene in S. cerevisiae; An efficient and economical yeast expressing system(K601-p416 system) which is suitable for the expression of FAD was built.