Xiangying Meng

Cantharidin reverses multidrug resistance of human hepatoma HepG2/ADM cells via down-regulation of P-glycoprotein expression.

Multidrug resistance (MDR) is a serious obstacle encountered in cancer treatment. In this study, we established an in vitro multiple drug resistant HepG2 cell line (HepG2/ADM), and characterized its MDR. This model was used to screen potential candidate chemosensitisers from over 200 purified naturally occurring compounds extracted from plants and animals. Cantharidin was found to have a significant reversal on MDR in our model. Further, our results showed that Cantharidin could significantly inhibit P-gp (P-glycoprotein) expression, mRNA transcription, as well as MDR1 promoter activity. These results suggest that Cantharidin is a novel and potent MDR reversal agent and may be a potential adjunctive agent for tumor chemotherapy.

Basic FGF downregulates TSP50 expression via the ERK/Sp1 pathway

Previous studies demonstrated that the expression of testes‐specific protease 50 (TSP50) was increased in breast cancer cells and that overexpression of TSP50 can promote tumorigenesis. Thus, it is important to identify the regulatory mechanisms of TSP50 for tumor therapy. In this study, we elucidated the mechanism underlying TSP50 downregulation by basic fibroblast growth factor (bFGF). We used MDA‐MB‐231 and HEK293T cell lines to address this issue. RT‐PCR and promoter activity assays indicated that bFGF downregulates TSP50 expression via transcriptional activation. We next investigated the signaling pathway that mediated the effect of bFGF on TSP50 transcription, and identified that bFGF induced the phosphorylation of ERK and Sp1. An ERK inhibitor suppressed Sp1 phosphorylation and bFGF‐reduced TSP50 expression at the mRNA level. In addition, the dominant negative (DN) mutants of ERK and Sp1 both suppressed the reduction of TSP50 by bFGF. Deletion and mutation analyses indicated that the Sp1 site, located within the +237/+239 region of the human TSP50 promoter, is the major responsive element for bFGF. Taken together, our results strongly suggest that bFGF mediates TSP50 downregulation by ERK activation, leading to the phosphorylation of Sp1 in this process. J. Cell. Biochem. 111: 75–81, 2010.

Components of Essential Oils in Different Parts of Daucus carota L. var. sativa Hoffm

The components of the essential oils obtained from different parts of Daucus carota L. var. sativa Hoffm were analyzed. The percentages of the essential oils extracted are 0.27% (ml/100 g material) for the flowers, 0.07% for the stems and leaves and 0.01% for the roots. Fifty-four, Sixty-six and Thirty-three compounds were, respectively, separated and identified from the flowers, the stems and leaves and the roots, among which unsaturated alkene compounds are thirty-nine, thirty-nine and twenty-one, respectively, accounting in turn for up to 90.21%, 90.49% and 72.65% of the total essential oils. Because alkene compounds have double bonds that are easily oxidized, it can be inferred that the components of the essential oils in the different parts of Daucus carota L. var. sativa Hoffm should show an activity of the anti-formation of free radicals to some extent.

Genetic and Phytochemical Analysis of Armillaria mellea by RAPD, ISSR, and HPLC

Abstract Molecular genetic and phytochemical methods were combined to investigate 17 Chinese strains of Armillaria mellea. Ten random amplified polymorphic DNA (RAPD) primers yielded 106 bands, of which 94 were polymorphic; 80 out of the 84 bands produced by nine inter-simple sequence repeats (ISSR) markers were polymorphic. Contents of water-soluble constituents of mycelia were characterized by high-performance liquid-chromatography–diode array detection (HPLC-DAD) analyses. Cluster analyses of the genetic and phytochemical variation revealed that Armillaria mellea exhibited four chemotypes and four clusters from phytochemical contents. Phylogenetic groupings displayed in tree plots calculated from the RAPD and ISSR data matrix correlated with the geographical origin of the fungi materials. Genetic profiles partially correlated with the chemotypes. Phytochemical contents mainly correlated with the strains. A method based on RAPD and HPLC is described to establish genetic and chemical quality control of Armillaria mellea simultaneously. Ten RAPD primers and a coefficient of >0.95 can be used in authentication of Armillaria mellea. The fingerprints obtained with HPLC consist of 22 common peaks within 45 min. This method could be readily utilized as a quality-control method for pharmaceutical-grade Armillaria mellea.

Preparation of Polyclonal Antibodies Against Testis-specific Protease 50 and Characterization of Antibody Specificity

Testis-specific protease 50 (TSP50) is a testis-specific oncogene, which is abnormally activated in most tested patients with breast cancer. This property makes it an attractive molecular marker and a promising target for the diagnosis and therapy of breast cancer. In order to obtain the protective and specific polyclonal antibodies for further research, TSP50 cDNA was amplified by RT-PCR from normal human testicular tissue, and inserted into eukaryotic expression vector pcDNA3. 1. Rabbit anti-TSP50 polyclonal antibodies were prepared by means of intramuscular injection of pcDNA3. 1-TSP50 into the rabbits. Titers of the anti-sera were measured by ELISA and Western blotting with the E. coli cell lysate containing the induced GST-TSP50 fusion protein as an antigen. In addition, we examined the expression of TSP50 in both breast cancer cell line MCF-7 and breast cancer tissue by immunofluorescent and immunohistochemistry analysis.

Isolation and Characterization of Proteins Interacting with Activin Type II Receptors

Regulation of the number of activin receptors that are present in the cell membrane plays a key role in the modulation of cellular responses to activin. In order to find the regulators, a novel protein ARIPzip, interacting with activin type II receptors, was searched and identified by using yeast two-hybrid screening. ARIPzip is a splicing variant of ARIP2. This has been discussed previously. ARIPzip can specifically interact with ActR II A, and is widely distributed in mouse tissues. Overexpression of ARIPzip can cause the activin-induced transcriptional activities to increase in a dose-dependent manner while the overexpression of ARIP2 can decrease these activities. These data suggest that the C-terminal regions of ARIP2 and ARIPzip are involved in the regulation of activin signaling.

Mutation Analysis of AVPR2 and AQP2 Gene in Chinese Patients with Congenital Nephrogenic Diabetes Insipidus

Abstract To detect mutations of the aquaporin 2 gene(AQP2) and the arginine vasopressin V2 receptor gene(AVPR2) of Chinese congenital nephrogenic diabetes insipidus, and to establish the foundation for further studying the emergence mechanism of the disease and clinical diagnosis, all the exons and part of introns of AQP2 and AVPR2 genes were amplified with intronic primers, using genomic DNA extracted from three patients with congenital nephrogenic diabetes insipidus and two mothers as template, PCR product was ligated into a T-vector and then sequenced. The result was compared with the database sequence to identify the mutable sites via a BLAST search, the incidence of every mutation was analyzed, and the putative transcription factor binding sites that maybe disturbed were analyzed by MAPPER. Mutation g.1394A>G in exon 3 of AVPR2 was detected in all the subjects, g.861C>T(S167L) in exon 2 of AVPR2 and IVS1+3G>A in intron of AQP2 were detected, respectively, in two patients, and c.836A>C in 3′ untranslated region of AQP2 was detected in two patients and one mother. Four mutations were identified. g.1394A>G of AVPR2 and c.836A>C of AQP2 have high incidence in patients with nephrogenic diabetes insipidus. Detection on the two sites may become auxiliary diagnosis index of congenital nephrogenic diabetes insipidus.

Construction and Characterization of a cDNA Expression Library for the North American Ginseng Root Tissues

The root of Panax ginseng plant undergoes a specific developmental process to become a biosynthesis and accumulation tissue for ginsenosides. To identify and analyze genes involved in the biosynthesis of ginsenoside, we constructed and characterized a full-length cDNA library for 6-year-old North American ginseng. The titer of primary cDNA library is 1. 2 × 10 6 pfu/mL, the titer of amplified library is 2. 6 × 10 10 pfu/mL and the rate of recombinant is above 86%. The insert size ranges from 0. 3 to 2. 0 kb. Sequencing results show that 18 of 58 genes are high homologous to the genes (GBR5, GBR3 and GBR1) known in GenBank, which are involved in biosynthesis of ginsenoside in North American ginseng plant; 16 of 58 genes are novel genes. The full-length cDNA library of North American ginseng root tissues is essential for the cloning of genes known and it is also an initial key for the screening and cloning of new genes.

Synergistic Effects of Activin A and Fibroblast Growth Factor 2 in the Modulation of Insulin Expression

Diabetes is the most prevalent and serious metabolic disease, and the number of diabetic patients worldwide is increasing. The reduction of insulin biosynthes is in pancreatic p-cells is closely associated with the onset and progression of diabetes, therefore, it is important to search for ways to induce insulin-producing cells in non-@cells. In the present study, it has been reported that activin A and a basic fibroblast growth factor 2( FGF2) , can synergistically increase the insulin mRNA level, in both mouse El4 striatal primary cell cultures and the hippocampal neuronal cell line HT22. Activin A and FGF2 can jointly stimulate the nuclear translocation of Smad3 and specifically activate ERK1/2. It is interesting to note that a specific inhibitor for MEK, U0126, can efficiently block the induction of an insulin promoter activity by activin A and FGF2. This indicates that activin A collaborates with FGF2, giving a signal to induce the insulin gene through selective activation of the ERK-type MAP kinase and Smad3 in mouse striatal and Hl2.2 cells. These data suggest that activin A may act in concert with FGF2 for the development of insulin -positive neurons.