Comparison of the substrate specificity of the two bacterial desulfurization systems

Using cell-free extracts of a desulfurizing mesophile, Rhodococcus erythropolis KA2-5-1 (the Dsz system) and Escherichia coli JM109, which possesses the desulfurizing genes of a thermophile Paenibacillus sp. A11-2 (the Tds system), the reactivity of desulfurizing enzymes toward 4,6-dialkyl dibenzothiophenes (4,6-dialkyl DBTs) and 7-alkyl benzothiophenes (7-alkyl BTs) was investigated. Both systems desulfurized all the 4,6-dialkyl DBTs, except 4,6-dibutyl DBT. Although some alkylated BTs were degraded by the Dsz system, no desulfurized compounds were detected. The reactivity of the Tds system toward alkylated BTs was higher than that of DBT. In contrast to the Dsz system, the Tds system yielded desulfurized compounds from all of the alkylated BTs examined.     

Betulinic and oleanolic acids isolated from Forsythia suspensa Vahl inhibit urease activity of Helicobacter pylori

Sixteen medicinal herbs were selected from a database on traditional herbal materials as well as literature on Korean plant resources. Then ethanol (70%, v/v) extracts of these herbs were tested for inhibition of the urease activity of Helicobacter pylori. The urease activity of H. pylori was strongly (82%) inhibited by extract of Forsythia suspensa Vahl. Active compounds in extract of Forsythia suspensa Vahl were first separated by batch mode solvent extraction, followed by purification by silica gel and octadecyl silica gel column chromatography using solvents of different polarity. According to NMR analysis of the last chromatographic fraction, we identified the presence of betulinic acid and oleanolic acid, which are known to have anti-inflammatory, anti-cancer, and anti-HIV viral activities.     

Induction of Betalain Pigmentation in Hairy Roots of Red Beet under Different Radiation Sources

The effects of aluminum on lipid peroxidation and activities of antioxidative enzymes were investigated in detached rice leaves treated with 0 to 5 mM AlCl₃ at pH 4.0 in the light. AlCl₃ enhanced the content of malondialdehyde but not the content of H₂O₂. Superoxide dismutase activity was reduced by AlCl₃, while catalase and glutathione reductase activities were increased. Peroxidase and ascorbate peroxidase activities were increased only after prolonged treatment, when toxicity occurred. The results give evidence that Al treatment caused oxidative stress and in turn, it caused lipid peroxidation.     

Down-regulation of trypsinogen expression is associated with growth retardation in alpha1,6-fucosyltransferase-deficient mice: attenuation of proteinase-activated receptor 2 activity

Alpha1,6-fucosyltransferase (Fut8) plays important roles inphysiological and pathological conditions. Fut8-deficient (Fut8^–/–)mice exhibit growth retardation, earlier postnatal death, andemphysema-like phenotype. To investigate the underlying molecularmechanism by which growth retardation occurs, we examined themRNA expression levels of Fut8^–/– embryos (18.5days postcoitum [dpc]) using a cDNA microarray. The DNA microarrayand real-time polymerase chain reaction (PCR) analysis showedthat a group of genes, including trypsinogens 4, 7, 8, 11, 16,and 20, were down-regulated in Fut8^–/– embryos.Consistently, the expression of trypsinogen proteins was foundto be lower in Fut8^–/– mice in the duodenum, smallintestine, and pancreas. Trypsin, an active form of trypsinogen,regulates cell growth through a G-protein-coupled receptor,the proteinase-activated receptor 2 (PAR-2). In a cell culturesystem, a Fut8 knockdown mouse pancreatic acinar cell carcinoma,TGP49-Fut8-KDs, showed decreased growth rate, similar to thatseen in Fut8^–/– mice, and the decreased growth ratewas rescued by the application of the PAR-2-activating peptide(SLIGRL-NH₂). Moreover, epidermal growth factor (EGF)-inducedreceptor phosphorylation was attenuated in TGP49-Fut8-KDs, whichwas highly associated with a reduction of trypsinogens mRNAlevels. The addition of exogenous EGF recovered c-fos, c-jun,and trypsinogen mRNA expression in TGP49-Fut8-KDs. Again, theEGF-induced up-regulation of c-fos and c-jun mRNA expressionwas significantly blocked by the protein kinase C (PKC) inhibitor.Our findings clearly demonstrate a relationship between Fut8and the regulation of EGF receptor (EGFR)-trypsin-PAR-2 pathwayin controlling cell growth and that the EGFR-trypsin-PAR-2 pathwayis suppressed in TGP49-Fut8-KDs as well as in Fut8^–/–mice.     

Crankshaft motions of the polypeptide backbone in molecular dynamics simulations of human type-α transforming growth factor

Summary Order parameters for the backbone N–H and C–H bond vectors have been calculated from a 150 ps molecular dynamics (MD) simulation of human type- transforming growth factor in H₂O solvent. Two kinds of crankshaft motions of the polypeptide backbone are observed in this MD trajectory. The first involves small-amplitude rocking of the rigid peptide bond due to correlated changes in the backbone dihedral angles _i–1 and _i. These high-frequency librational crankshaft motions are correlated with systematically smaller values of motional order parameters for backbone N–H bond vectors compared to C–H bond vectors. In addition, infrequent crankshaft flips of the peptide bond from one local minimum to another are observed for several amino acid residues. These MD simulations demonstrate that comparisons of N–H and C–H order parameters provide a useful approach for identifying crank-shaft librational motions in proteins.     

A single residue mutation of 5-enoylpyruvylshikimate-3-phosphate synthase in Pseudomonas stutzeri enhances resistance to the herbicide glyphosate

A novel class II 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) was identified from Pseudomonas stutzeri A1501 by complementation of an Escherichia coli auxotrophic aroA mutant. The single amino acid substitution of serine (Ser) for asparagine (Asn)-130 of the A1501 EPSPS enhanced resistance to 200 mM glyphosate. The mutated EPSPS had a 2.5-fold increase for IC(50) [glyphosate] value, a 2-fold increase for K (i) [glyphosate] value, but a K (m) [PEP] value similar to that of wild type. The effect of the single residue mutation on glyphosate resistance was also analyzed using a computer-based three-dimensional model.     

Improvement of pristinamycin production by genome shuffling and medium optimization for Streptomyces pristinaespiralis

To isolate an improved pristinamycin producing strain of Streptomyces pristinaespiralis, the technique of Genome shuffling was used which resulted in a high-yield recombinant G 3-56 strain. Strain G 3-56 yielded 322 ± 17 mg/L of pristinamycin which was 11.4-fold higher than that of the initial strain and 3.7-fold higher than strain UN-78 which previously had the highest yield of pristinamycin. The genetic characteristics of the recombinant G 3-56 strain was stable as revealed by our subculture experiments. The optimal production medium was determined using the orthogonal matrix method. Under the optimal medium conditions, the maximum yield of pristinamycin was 412 mg/L with about 1.24-fold higher than the original medium.     

Gene expression profile analysis in astaxanthin-induced Haematococcus pluvialis using a cDNA microarray

The unicellular green alga Haematococcus pluvialis (Volvocales) is known for the ketocarotenoid astaxanthin (3, 3′-dihydroxy-β, β-carotene-4, 4′-dione) accumulation, which is induced under unfavorable culture conditions. In this work, we used cDNA microarray analysis to screen differentially expressed genes in H. pluvialis under astaxanthin-inductive culture conditions, such as combination of cell exposure to high irradiance and nutrient deprivation. Among the 965 genes in the cDNA array, there are 144 genes exhibiting differential expression (twofold changes) under these conditions. A significant decrease in the expression of photosynthesis-related genes was shown in astaxanthin-accumulating cells (red cells). Defense- or stress-related genes and signal transduction genes were also induced in the red cells. A comparison of microarray and real-time PCR analysis showed good correlation between the differentially expressed genes by the two methods. Our results indicate that the cDNA microarray approach, as employed in this work, can be relied upon and used to monitor gene expression profiles in H. pluvialis. In addition, the genes that were differentially expressed during astaxanthin induction are suitable candidates for further study and can be used as tools for dissecting the molecular mechanism of this unique pigment accumulation process in the green alga H. pluvialis. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Expression of Discoidin Domain Receptor 2 (DDR2) Extracellular Domain in Pichia Pastoris and Functional Analysis in Synovial Fibroblasts and NIT3T3 Cells

Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, identified as a ligand for DDR2, up-regulates matrix metallloproteinase 1 (MMP-1) and MMP-2 expression in cellular matrix. To investigate the roles of DDR2 in destruction of cartilage in rheumatoid arthritis (RA) and tumor metastasis, we tried to express extracellular domain of DDR2 fused with a His tag to increase protein solubility and facilitate purification (without signal peptide and transmembrane domain, designated DR) in Pichia pastoris, purify the expressed protein, and characterize its function, for purpose of future application as a specific DDR2 antagonist. Two clones of relative high expression of His-DR were obtained, After purification by a Ni-NTA (nitric-tri-acetic acid) chromatographic column, soluble fused His-DR over 90% purity were obtained. Competitive binding inhibition assay demonstrated that expressed His-DR could block the binding of DDR2 and natural DDR2 receptors on NIT3T3 and synovial cell surfaces. Results of RT-PCR, Western blotting, and gelatinase zymography showed that His-DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIT3T3 cells and RA synoviocytes stimulated by collagen II. For MMP-1, the inhibitory effect was displayed at the levels of mRNA and protein, whereas for MMP-2 it was demonstrated at the level of protein physiological activity. All these findings suggested that the fused expressed His-DR inhibited the activity of natural DDR2, and relevant MMP-1 and MMP-2 expression in synoviocytes and NIH3T3 cells provoked by collagen II. Wei Zhang and Tianbing Ding equally contributed to this work.     

Genetic spatial clustering: significant implications for conservation of wild soybean (Glycine soja: Fabaceae)

Knowledge of spatial patterns of genetic variation within populations of wild relative species has significant implications with respect to sampling strategies for ex situ and in situ conservation. To study spatial genetic structure of wild soybean (Glycine soja Sieb. et Zucc.) at the fine scale, three natural populations in northern China were analyzed using inter-simple sequence repeat (ISSR) fingerprints for estimating kinship coefficients. A regression analysis of kinship coefficients against spatial distances revealed that individuals occurring close together tended to be more genetically related. The Sp statistic further indicated a comparable spatial pattern among the three wild soybean populations with similar Sp values (mean = 0.0734, varied from 0.0645 to 0.0943) detected across the three populations. Genetic patches were on average ca. 20 m in size, and the effective neighborhood sizes varied between 10 and 15 m. The spatial genetic structure evident in the wild soybean populations may be attributed to the restricted seed dispersal and predominant inbreeding mating system of this species. The detection of family structure in the populations of wild soybean has a significant implication for the effective conservation of the important genetic resources.