The Synergistic Effect of Serine with Selenocompounds on the Expression of SelP and GPx in HepG2 Cells.

We explored the synergistic effect of serine combined with several selenocompounds or used alone on the expression of selenoprotein P (SelP) and glutathione peroxidase (GPx) in this study. We first compared the SelP and GPx expression difference between HepG2 and Hela cells treated with serine and finally chose HepG2 as experimental cell. In the serine-used-alone experiment, three kinds of selenium nutritional models (low-, adequate-, and high-selenium) were established and serine was 10 times gradient diluted (0.01 to 100 µmol/L). In the combined experiment, the selenocompound doses were set as 0.01, 0.1, and 1 µmol Se/L and serine was set according to its molar ratio with the selenocompounds. We found that SelP and GPx concentrations in the low-, adequate-, and high-selenium models increased following with serine dose. When the concentration of sodium selenite and SeMet was 1 µmol Se/L while MeSeCys was 0.1 and 1 µmol Se/L, SelP concentrations for serine combined with selenocompounds groups were significantly higher than that of selenocompounds used alone. When the concentration of sodium selenite was 0.1 µmol Se/L, SeMet was 0.1 and 1 µmol Se/L while MeSeCys was 0.01 and 1 µmol Se/L, GPx concentrations for serine combined with selenocompounds groups were significantly higher than that of selenocompounds used alone. Our preliminary result indicated the beneficial effect of serine on the expression of SelP and GPx, which suggested that it might be a candidate for combined selenium supplement.

Suppression effects of betaine-enriched spinach on hyperhomocysteinemia induced by guanidinoacetic acid and choline deficiency in rats.

Betaine is an important natural component of rich food sources, especially spinach. Rats were fed diets with betaine or spinach powder at the same level of betaine for 10 days to investigate the dose-dependent effects of spinach powder supplementation on hyperhomocysteinemia induced by guanidinoacetic acid (GAA) addition and choline deprivation. The GAA-induced hyperhomocysteinemia in rats fed 25% casein diet (25 C) was significantly suppressed by supplementation with betaine or spinach, and it was completely suppressed by taking 11.0% spinach supplementation. The choline deprivation-induced enhancement of plasma homocysteine concentration in rats fed 25% soybean protein diet (25S) was markedly suppressed by 3.82% spinach. Supplementation with betaine or spinach partially prevented the effects of GAA on hepatic concentrations of methionine metabolites. The decrease in activity of betaine-homocysteine S-methyltransferase (BHMT) and cystathionine ß-synthase (CBS) in GAA-induced hyperhomocysteinemia was recovered by supplementation with betaine or spinach. Supplementation with betaine or spinach did not affect BHMT activity, whereas it partially restored CBS activity in choline-deprived 25S. The results indicated that betaine or spinach could completely suppress the hyperhomocysteinemia induced by choline deficiency resulting from stimulating the homocysteine removal by both remethylation and cystathionine formation.

EBP50 exerts tumor suppressor activity by promoting cell apoptosis and retarding extracellular signal-regulated kinase activity.

The expression of Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) and the intragenic mutation of the ebp50 gene have been reported to correlate with human breast cancer development, but the exact impacts on breast cancer development and its molecular mechanism are not fully understood. In this study, we investigate the potential function of EBP50 through over-expression in the breast cancer cell line, MDA-MB-231, which has low EBP50 protein expression levels. The effects of EBP50 over-expression on cellular proliferation, anchorage-independent growth and apoptosis were examined. In addition, the activity of extracellular signal-regulated kinase (ERK) was also determined. Our results show that a decrease of cellular proliferation and attenuation of colony-forming ability were evident in MDA-MB-231 cells stably transfected with an EBP50 expressing plasmid (EBP-231) when compared with control cells. There was also a statistically significant increase in spontaneous apoptosis in EBP-231 cells accompanied by an attenuation in ERK activity. Altogether, our results suggest that restoring EBP50 expression could suppress breast cancer cell proliferation by promoting cell apoptosis and inhibiting ERK activity, and that EBP50 may be a target for development of diagnostics and therapeutics in breast cancer.

[Construction of VEGF recombinant plasmid pcDNA/V and its expression in model rats with acute myocardial ischemia].

The cDNA encoding human Vascular Endothelial Growth Factor 165 (VEGF165) was amplified using RT-PCR from human tonsil tissue and cloned into eukaryotic expression vector pcDNA3.1 (+). The recombinant plasmid pcDNA/V was transferred into 293 cells mediated by liposome and the cells stably expressing VEGF were selected under the pressure of G418. ELISA and Western blotting demonstrated that the eukaryotic expression vector pcDNA/V was successfully constructed and its corresponding protein could be expressed efficiently in vitro. Chick Charioallantoic Membrane (CAM) bioassay showed that recombinant protein has biological activity of hVEGF. Model rats with acute myocardial ischemia were used to further study the expression of VEGFin vivo. The model rats were divided randomly into three groups: control group, pcDNA3.1 (+) group and pcDNA/V group. 50microL naked plasmid DNA or saline was intramyocardially injected at three sites into the border zone of infarction. The hearts of rats were excised and fixed histologically, then the infarction sizes were studied by immunohistochemical staining and electron microscope after four weeks. Immunohistochemical staining for VEGF appeared to be negative in control and pcDNA3.1 (+) groups. In pcDNA/V group, myocardial cells in infarction border zone showed positive staining for VEGF in cytoplasm. Ultrastructural anaylsis showed that there were visible hyperplasia of vascular endothilium in pcDNA/V group. The control and pcDNA3.1 (+) groups showed less capillary hyperplasia. In this study, VEGF165 gene was successfully cloned and its protein expressed in vitro and in vivo was of bioactivity, which provides a basis for the further study of biological functions of human VEGF.