Production of glutamine synthetase in <Emphasis Type="Italic">Escherichia coli</Emphasis> using SUMO fusion partner and application to <Emphasis Type="SmallCaps">l</Emphasis>-glutamine synthesis

l-glutamine (Gln) is an important conditionally necessary amino acid in human body and potential demand in food or medicine industry is expected. High efficiency of l-Gln production by coupling genetic engineered bacterial glutamine synthetase (GS) with yeast alcoholic fermentation system has been developed. We report here first the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Bacillus subtilis GS. In order to obtain GS with high Gln-forming activity, safety and low cost for food and pharmaceutics industry, 0.1% (w/v) lactose was selected as inducer. The fusion protein was expressed in totally soluble form in E. coli, and expression was verified by SDS–PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel nitrilo-triacetic acid (Ni–NTA) resin chromatography with a yield of 625 mg per liter fermentation culture. After the SUMO/GS fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni–NTA column. Finally, about 121 mg recombinant GS was obtained from 1 l fermentation culture with no less than 96% purity. The recombinant purified GS showed great transferase activity (23 U/mg), with 25 U recombinant GS in a 50 ml reaction system, a biosynthesis yield of 27.5 g/l l-Gln was detected by high pressure liquid chromatography (HPLC) or thin-layer chromatography. Thus, the application of SUMO technology to the expression and purification of GS potentially could be employed for the industrial production of l-Gln.     

Polysialylation promotes neural cell adhesion molecule-mediated cell migration in a fibroblast growth factor receptor-dependent manner, but independent of adhesion capability

Polysialic acid (PSA), a carbohydrate polymer mainly present in the neural cell adhesion molecule (NCAM), promotes neural plasticity; however, its mode of action in tumor malignancy remains largely unknown. In this study, we investigated the influence of polysialylation on cell migration. PSA consistently promoted cell migration on different extracellular matrices (ECMs) but differentially affected cell adhesion. All of these actions were reversed by endo-N-acetylneuraminidase treatment, and PSA-driven migration was inhibited by the specific fibroblast growth factor receptor (FGFR) inhibitor Su5402. Consistent with this latter observation, PSA-stimulated migration on different ECMs was paralleled by activation of the FGFR and its downstream signaling components, PLC-γ, focal adhesion kinase and extracellular signal-regulated kinase 1/2. In contrast, the pattern of p59(fyn) activation correlated with differential adhesion to different ECMs. Collectively, these results indicate that PSA-conjugated NCAM potentiates signal transduction by the FGFR pathway and thereby enhances cell migration independent of adhesion capability, providing additional insights into the role of PSA in cancer development.     

A specific role for Arabidopsis TRAPPII in post-Golgi trafficking that is crucial for cytokinesis and cell polarity

Cytokinesis and cell polarity are supported by membrane trafficking from the trans-Golgi network (TGN), but the molecular mechanisms that promote membrane trafficking from the TGN are poorly defined in plant cells. Here we show that TRAPPII in Arabidopsis regulates the post-Golgi trafficking that is crucial for assembly of the cell plate and cell polarity. Disruptions of AtTRS120 or AtTRS130, two genes encoding two key subunits of TRAPPII, result in defective cytokinesis and cell polarity in embryogenesis and seedling development. In attrs120 and attrs130, the organization and trafficking in the endoplasmic reticulum (ER)-Golgi interface are normal. However, post-Golgi trafficking to the cell plate and to the cell wall, but not to the vacuole, is impaired. Furthermore, TRAPPII is required for the selective transport of PIN2, but not PIN1, to the plasma membrane. We revealed that AtTRS130 is co-localized with RAB-A1c. Expression of constitutively active RAB-A1c partially rescues attrs130. RAB-A1c, which resides at the TGN, is delocalized to the cytosol in attrs130. We propose that TRAPPII in Arabidopsis acts upstream of Rab-A GTPases in post-Golgi membrane trafficking in plant cells.     

Cell-penetrating peptides derived from viral capsid proteins

Cell-penetrating peptides (CPP) can translocate across the cell membrane and have been extensively studied for the delivery of proteins, nucleic acids, and therapeutics in mammalian cells. However, characterizations of CPP in plants have only recently been initiated. We showed that the intact virion and a recombinant capsid protein (CaP) from a plant-infecting nonenveloped icosahedral RNA virus, Brome mosaic virus (BMV), can penetrate the membranes of plant protoplasts but are trapped by the extracellular matrix. Furthermore, a 22-residue peptide derived from the N-terminal region of the CaP (CPNT) can enter barley protoplasts and cells of intact barley and Arabidopsis roots. An inhibitor of the macropinocytosis reduced CPNT entry, while treatment with NiCl(2) changed the cellular localization of CPNT. CPNT increased uptake of the green flourescent protein (GFP) into the cell when covalently fused to GFP or when present in trans of GFP. The BMV CPNT overlaps with the sequence known to bind BMV RNA, and it can deliver BMV RNAs into cells, resulting in viral replication, as well as deliver double-stranded RNAs that can induce gene silencing.     

Simulation study on water quality based on sediment release flume experiment in Lake Taihu, China

Sediments have a significant influence on the overlying water, and nutrient release from sediments is an important source for lake eutrophication, particularly in shallow lakes. Sediment resuspension is primarily driven by wind-induced currents. In this research, the correlation between release rate of suspended sediment and flow velocity was studied, and an experiment on hydrodynamic forces was conducted in a rectangle flume using water and sediments collected from three sites in Lake Taihu, a eutrophic lake in China. It was shown that the starting velocities of sediment in Lake Taihu at three different incipient standards gained from the experiment were 15, 30, and 40 cm s⁻¹ and the release rate of suspended sediment could reach up to 643.4, 5377.1, and 13980.5 g m⁻² d⁻¹, respectively. Based on the experiment, a water quantity and quality numerical model of wind-induced current with sediment pollution for Lake Taihu was developed. The model was calibrated and validated by applying it to the study of the water quality of Lake Taihu. The calculated values were generally in good agreement with field observations, which indicated that the developed model could represent the dynamics of sediment resuspension to a certain extent. This study provides a new approach and a practical tool for planning and management policy and operations to protect the water quality and ecosystems of shallow lakes.     

Identification of novel α-gliadin genes

Ten novel α-gliadin genes (Gli-ta, Gli-turg1, Gli-turg2, Gli-turg3, Gli-turg4, Gli-turg5, Gli-turg6, Gli-cs1, Gli-cs2, and Gli-cs3) with unique characteristics were isolated from wheat (Triticum aestivumL.), among which Gli-cs1, Gli-cs2, Gli-cs3, and Gli-turg6 were pseudogenes. Gli-cs3 and nine other sequences were much larger and smaller, respectively, than the typical α-gliadins. This variation was caused by insertion or deletion of the unique domain I and a polyglutamine region, possibly the result of illegitimate recombination. Consequently, Gli-cs3 contained 10 cysteine residues, whereas there were 2 cysteine residues only in the other nine sequences. Gli-ta/Gli-ta-like α-gliadin genes are normally expressed during the development of seeds. SDS-PAGE analysis showed that in-vitro-expressed Gli-ta could form intermolecular disulphide bonds and could be chain extenders. A protein band similar in size to Gli-ta has been observed in seed extracts, and mass spectrometry results confirm that the band contains small molecular mass α-gliadins, which is a characteristic of the novel α-gliadins. Mass spectrometry results also indicated that the two cysteine residues of Gli-ta/Gli-ta-like proteins participated in the formation of intermolecular disulphide bonds in vivo.     

Hypoglycemic activities of A- and B-type procyanidin oligomer-rich extracts from different Cinnamon barks

Procyanidin oligomers in Cinnamon are thought to be responsible for the biological activity in the treatment of diabetes mellitus (DM). To clarify types of procyanidin oligomers in different Cinnamon species and investigate their different effects, the present study investigated procyanidin oligomers in polyphenolic oligomer-rich extracts of three Cinnamon samples by LC-MS methods, and their hypoglycemic activities were detected in vivo and in vitro. The results showed that two of the three samples from Cinnamomum cassia were rich in B-type procyanidin oligomers, and the other sample was rich in A-type procyanidin oligomers. The Cinnamon extracts were administered at doses of 200 and 300 mg/kg body wt. in high-fat diet-fed and low-dose streptozotocin (STZ)-induced diabetic mice for 14 days. The results showed that blood glucose concentrations were significantly decreased in all Cinnamon extract groups compared with the control group (p < 0.05). Administration of the Cinnamon extracts significantly increased the consumption of extracellular glucose in insulin-resistant HepG2 cells and normal HepG2 cells compared with the control group. These results suggest that both A- and B-type procyanidin oligomers in different Cinnamon species have hypoglycemic activities and may improve insulin sensitivity in type 2 DM.     

Development of benzothiazole 'click-on' fluorogenic dyes

‘Click-on’ fluorogenic reaction: a non-fluorescent benzothiazole with an electron-deficient alkyne group at 2-position reacts with azide containing molecules could form fluorescent adducts.     

Synthesis and acrosin inhibitory activity of substituted 4-amino-N-(diaminomethylene) benzenesulfonamide derivatives

A series of new substituted 4-amino-N-(diaminomethylene) benzenesulfonamides were synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds showed potent acrosin inhibitory activities with compounds 4o and 4p being significantly more potent than the control compound N-alpha-tosyl-l-lysyl-chloromethyl-ketone (TLCK). The compounds provide a new scaffold for the development of acrosin inhibitory agents.     

EGID: an ensemble algorithm for improved genomic island detection in genomic sequences

Genomic islands (GIs) are genomic regions that are originally transferred from other organisms. The detection of genomic islands in genomes can lead to many applications in industrial, medical and environmental contexts. Existing computational tools for GI detection suffer either low recall or low precision, thus leaving the room for improvement. In this paper, we report the development of our Ensemble algorithm for Genomic Island Detection (EGID). EGID utilizes the prediction results of existing computational tools, filters and generates consensus prediction results. Performance comparisons between our ensemble algorithm and existing programs have shown that our ensemble algorithm is better than any other program. EGID was implemented in Java, and was compiled and executed on Linux operating systems. EGID is freely available at http://www5.esu.edu/cpsc/bioinfo/software/EGID.