Jihui Wang

Roles of tetrahydrobiopterin in promoting tumor angiogenesis.

Nitric oxide (NO), which is derived from endothelial NO synthase (eNOS), provides crucial signals for angiogenesis in the tumor microenvironment. Tetrahydrobiopterin (BH4) is an absolute requirement for eNOS activity. In this study, we investigated whether this activation is both maintained by a wild-type Ras/phosphatidylinositol 3-kinase (PI3K)/Akt-positive feedback loop in endothelial cells and affects tumor angiogenesis. We found that supplementation of BH4 (via the pterin salvage pathway with Sep) increased Akt/eNOS phosphorylation in both human eNOS-transfected COS-7 cells and endothelial cells concomitant with increases in NO production, cell proliferation, migration, and tube formation. This augmentation was abrogated by a PI3K inhibitor. Sepiapterin (Sep) also increased GTP-bound wild-type Ras and PI3K/Akt/eNOS activation, which was prevented by the eNOS inhibitor, Nω-Nitro-L-arginine methyl ester (L-NAME). Furthermore, expression of GTP cyclohydrolase I (the rate-limiting enzyme in de novo BH4 synthesis) under doxycycline control potentiated in vivo tumorigenesis, tumor cell proliferation, as well as angiogenesis. Conversely, both switching off GTP cyclohydrolase I expression as well as inhibiting its enzymatic activity significantly decreased eNOS expression and tumor vascularization. This study demonstrates an important role for BH4 synthesis in angiogenesis by the activation of eNOS for NO production, which is maintained by a PI3K/Akt-positive feedback loop through effects on wild-type Ras in endothelial cells. Our findings suggest that BH4 synthesis may be a rational target for antiangiogenesis therapy for tumors.

Induction of apoptosis by c9, t11-CLA in human endometrial cancer RL 95-2 cells via ERα-mediated pathway

Numerous studies have shown that conjugated linoleic acid (CLA) can inhibit cancer cells growth and induce apoptosis in vitro and in vivo. The aim of the present study was to investigate the effects of CLA, including cis9, trans11-conjugated linoleic acid (c9, t11-CLA) and trans10, cis12-conjugated linoleic acid (t10, c12-CLA), on apoptosis of human endometrial cancer RL 95-2 cells and its related mechanisms. The MTT analysis was used to evaluate the effect of CLA isomers on the viability of endometrial cancer RL 95-2 cells. We then estimated the apoptosis by Morphological observation and Annexin V-FITC/PI staining and flow cytometry. We also used Western blot analysis to assess the expression of caspase-3, Bax, Bcl-2 proteins and the activation of Akt/p-Akt and ERα/p-ERα. Propylpyrazole-triol (PPT), a selective ERα agonist was used to confirm the induction of apoptosis by c9, t11 CLA may relate to ERα-mediated pathway. In CLA-treated RL 95-2 cells, we found that c9, t11-CLA inhibited viability and trigged apoptosis, as judged from nuclear morphology and flow cytometric analysis. The expression of caspase-3 and the ratio of Bax/Bcl-2 were significant increased, but no obvious change was observed about Akt and p-Akt in c9, t11-CLA-treated cells. However, the expression of total ERα level in RL 95-2 cells-treated with c9, t11-CLA was unchanged, while in the concentration of 80 mM, c9, t11-CLA down-regulated the protein expression level of p-ERα. Then PPT has the antagonistic action on growth inhibitory effect in RL 95-2 cells incubated with c9, t11-CLA. This study demonstrated that c9, t11- CLA could induce apoptosis in RL 95-2 cells, and may involve in ERα-mediated pathway. These results indicated that c9, t11- CLA could induce apoptosis of endometrial cancer cells and may be potential agents for the treatment of endometrial cancer.

Sensitivity enhancement of pH indicator and its application in the evaluation of fish freshness

The sensitivity of pH indicators is always confined in acidic solvents used during the immobilization because of the large deviation from their color transition points. By regulating the pH of indicators using organic acid or base, the color transition points of indicators could be readily achieved and the sensitivity could be greatly enhanced. In this study, bromophenol blue was selected as an example to interpret this improvement. And Hue, Saturation and Value (HSV) color model was applied to obtain digital color information of pH indicators. In addition, other two kinds of pH indicators were chosen and regulated to their color transition points as well. The resultant indicators together with the original ones were composed to a colorimetric sensor array and utilized to determine fish freshness. Compared with the original indicators, much higher sensitivity in the detection of fish freshness was obtained by the pH regulated indicators.

Induction of apoptosis and inhibition of invasion in choriocarcinoma JEG-3 cells by α-calendic acid and β-calendic acid.

Alfa-calendic acid and β-calendic acid, geometric and positional isomers of linolenic acid were previously shown to possess potent anticancer properties. In this study, we found that α-calendic acid and β-calendic acid could induce apoptosis and suppress invasion of human choriocarcinoma JEG-3 cells in vitro. Treatment with α-calendic acid and β-calendic acid significantly increased oxidative stress in human choriocarcinoma JEG-3 cells detected by the level of reactive oxygen species (ROS), lipid peroxidation production malondialdehyde (MDA), glutathione (GSH) and the effects of antioxidants NAC and α-tocopherol. Furthermore, oxidative stress activated the phosphorylation of p38MAPK. SB203580, a selective p38MAPK inhibitor, blocked the apoptosis induced by α-calendic acid and β-calendic acid by upregulating Bcl-2/Bax ratio and inhibition of the activation of Caspase-3 and Caspase-9. SB20350 also partially abrogated the cell invasion effects of α-calendic acid and β-calendic acid. These results suggested that α-calendic acid and β-calendic acid induced apoptosis and inhibited invasion in JEG-3 cells by activation of oxidative stress pathways and subsequent activation of P38MAPK.

Linoelaidic acid enhances adipogenic differentiation in adipose tissue-derived stromal cells through suppression of Wnt/β-catenin signaling pathway in vitro

Obesity has become a major health problem which is related with high-trans fatty acids diet. Adipogenic differentiation of adipose tissue-derived stromal cells (ADSCs) plays an important role in the development of adipose tissue. In order to determine the effect of trans fatty acids on adipogenic differentiation in ADSCs, cells were treated with linoelaidic acid, as well as linoleic acid and linolenic acid. We found that linoelaidic acid significantly increased the lipid droplet formation and triglyceride content compared with linoleic acid and linolenic acid. Linoelaidic acid also down-regulated the levels of β-catenin in cells and inhibited the accumulation of β-catenin in cell nuclei. Lithium chloride, an activator of Wnt/β-catenin pathway, antagonized the enhancement of linoelaidic acid on adipogenesis and up-regulated the levels of β-catenin in ADSCs. These results indicated that linoelaidic acid could enhance the adipogenic differentiation in ADSCs in vitro, which is partly due to the suppression of Wnt/β-catenin pathway.

Central carbon metabolism influences cellulase production in Bacillus licheniformis

Bacillus licheniformis that can produce cellulase including endo glucanase and glucosidase is an important industrial microbe for cellulose degradation. The purpose of this research was to assess the effect of endo glucanase gene bglC and glucosidase gene bglH on the central metabolic flux in B. licheniformis. bglC and bglH were knocked out using homologous recombination method, respectively, and the corresponding knockout strains were obtained for 13C metabolic flux analysis. A significant change was observed in metabolic fluxes after 13C metabolic flux ratio analysis. In both of the knockout strains, the increased fluxes of the pentose phosphate pathway and malic enzyme reaction enabled an elevated supply of NADPH which provided enough reducing power for the in vivo synthesis reactions. The fluxes through tricarboxylic acid cycle and anaplerotic reactions increased fast in the two knockout strains, which meant more energy generated. The changed fluxes in central carbon metabolism provided a holistic view of the physiological status in B. licheniformis and possible targets for further strain engineering.

Production of astaxanthin at moderate temperature in Xanthophyllomyces dendrorhous using a two-step process

In order to achieve continuous industrial production of astaxanthin in Xanthophyllomyces dendrorhous, a moderate temperature (25–37°C) fermentation process was needed. In this study, a two‐step process with a 20°C pre‐culture for 18 h and a 30°C culture for 30 h was performed to achieve the astaxanthin yields of 116.42 μg g−1 dry cell weight, which was lower than that in the normal process (20°C, 96 h). However, cell yield (YX/S) and product yield (YP/S) showed no significant differences between the two processes, suggesting that moderate temperature did not affect the productivity of astaxanthin. The transcriptional levels of genes involved in astaxanthin synthesis were compared in different culture times and a negative correlation between temperature and expression of carotenogenic genes was found. This work provided a potential method for continuous production of astaxanthin using X. dendrorhous at moderate temperature throughout the year.

The distribution and function characterization of the i type lysozyme from Apostichopus japonicus

ABSTRACT Lysozyme is a very important component of the innate immune system and a key molecule that protects against bacterial infection. Sea cucumber i‐type lysozyme (Aj‐iLys) has been shown to possess multiple functions. In this study, we investigated the function and characterization of Aj‐iLys in detail. Spatial distribution analysis showed that Aj‐iLys was constitutively expressed in all tested tissues, with dominant expression in the tentacles and respiratory trees. Challenge with the pathogen V. splendidus and LPS stimulation both significantly up‐regulated the mRNA expression of Aj‐iLys. More importantly, inhibition of Aj‐iLys expression by mRNA interference resulted in significant promotion of coelomocyte apoptosis during LPS challenge in vitro. The results indicated that Aj‐iLys serves as an important innate immunity factor and plays a key defense role during host‐pathogen interactions in sea cucumbers. From the radius of the antimicrobial zone, it was determined that the non‐fusion Aj‐iLys exerted a remarkable inhibitive effect on tested bacteria in vitro. Functional investigation revealed that Aj‐iLys also exhibited isopeptidase activity based on its ability to hydrolyze l‐Glutamic acid &ggr;‐(4‐nitroanilide) in vitro to produce p‐NA, which is an analogue of the isopeptide bond. The optimal catalytic conditions for the isopeptidase activity were 37 °C, pH 6.5, and the optimum ionic strength was about 0.050 mol/L. HIGHLIGHTSAj‐iLys participate and is up‐regulated in the innate immunity of sea cucumber during pathogenic stimulation.A very key regulatory role in the innate immunity of sea cucumber is proposed by the RNA interference for Aj‐iLys.Detecting the isopeptidase activity for Aj‐iLys and its characterization.