Expression of a lipase on the cell-surface of Escherichia coli using the OmpW anchoring motif and its application to enantioselective reactions

Microbial-surface display is the expression of proteins or peptides on the surface of cells by fusing an appropriate protein as an anchoring motif. Here, the outer membrane protein W (OmpW) was selected as a fusion partner for functional expression of Pseudomonas fluorescence SIK W1 lipase (TliA) on the cell-surface of Escherichia coli. Localization of the truncated OmpW-TliA fusion protein on the cell-surface was confirmed by immunoblotting and functional assay of lipase activity. Enantioselective hydrolysis of rac-phenylethyl butanoate by the displayed lipase resulted in optically active (R)-phenyl ethanol with 96 % enantiomeric excess and 44 % of conversion in 5 days. Thus, a small outer membrane protein OmpW, is a useful anchoring motif for displaying an active enzyme of ~50 kDa on the cell-surface and the surface-displayed lipase can be employed as an enantioselective biocatalyst in organic synthesis.     

The HEX1 Gene of Fusarium graminearum Is Required for Fungal Asexual Reproduction and Pathogenesis and for Efficient Viral RNA Accumulation of Fusarium graminearum Virus 1

The accumulation of viral RNA depends on many host cellular factors. The hexagonal peroxisome (Hex1) protein is a fungal protein that is highly expressed when the DK21 strain of Fusarium graminearum virus 1 (FgV1) infects its host, and Hex1 affects the accumulation of FgV1 RNA. The Hex1 protein is the major constituent of the Woronin body (WB), which is a peroxisome-derived electron-dense core organelle that seals the septal pore in response to hyphal wounding. To clarify the role of Hex1 and the WB in the relationship between FgV1 and Fusarium graminearum, we generated targeted gene deletion and overexpression mutants. Although neither HEX1 gene deletion nor overexpression substantially affected vegetative growth, both changes reduced the production of asexual spores and reduced virulence on wheat spikelets in the absence of FgV1 infection. However, the vegetative growth of deletion and overexpression mutants was increased and decreased, respectively, upon FgV1 infection compared to that of an FgV1-infected wild-type isolate. Viral RNA accumulation was significantly decreased in deletion mutants but was significantly increased in overexpression mutants compared to the viral RNA accumulation in the virus-infected wild-type control. Overall, these data indicate that the HEX1 gene plays a direct role in the asexual reproduction and virulence of F. graminearum and facilitates viral RNA accumulation in the FgV1-infected host fungus.     

Stimulation of Rg3 ginsenoside biosynthesis in ginseng hairy roots elicited by methyl jasmonate

It has been recognized that ginsenoside Rg3 is not naturally produced in ginseng although this ginsenoside can accumulate in red ginseng as the result of a thermal process. In order to determine whether or not Rg3 is synthesized in ginseng, hairy roots were treated with methyl jasmonate (MJ). From HPLC analysis, no peak for Rg3 was observed in the controls. However, Rg3 did accumulate in hairy roots that were MJ-treated for 7?days. Rg3 content was 0.42?mg/g (dry weight). To gain more insight into the effects of MJ on UDP-glucosyltransferase (UGT) activity, we attempted to evaluate ginsenoside Rg3 biosynthesis by UGT. A new peak for putative Rg3 was observed, which was confirmed by LC-MS/MS analysis. Our findings indicate that the proteins extracted from our hairy root lines can catalyze Rg3 from Rh2. This suggests that our ginseng hairy root lines possess Rg3 biosynthesis capacity.     

Effects of oral deoxynivalenol exposure on immune-related parameters in lymphoid organs and serum of mice vaccinated with porcine parvovirus vaccine

Mice were exposed to deoxynivalenol (DON) via drinking water at a concentration of 2 mg/L for 36 days. On day 8 of treatment, inactivated porcine parvovirus vaccine (PPV) was injected intraperitoneally. The relative and absolute weight of the spleen was significantly decreased in the DON-treated group (DON). Antibody titers to parvovirus in serum were 47.9?±?2.4 in the vaccination group (Vac), but 15.2?±?6.5 in the group treated with DON and vaccine (DON?+?Vac). The IgA and IgG was not different in the DON, Vac an,d DON?+?Vac groups. IgM was significantly lower only in the DON?+?Vac group. However IgE was significantly increased in the Vac and DON?+?Vac group, but no change was observed between the Vac and DON?+?Vac groups. The concentrations of IL-2, IL-4, GM-CSF, MCP-1 and Rantes in serum, and IL-1α in mesenteric lymph node and MIP-1β in spleen were significantly increased by DON treatment compared to control. The concentrations of IL-2, IL-5, IL-6, IL-9, IL-12, IL-13 and Rantes in thymus, of IL-2 in spleen, and of IL-1α, IL-1β, IL-3, IL-5, IL-10, IL-17, G-CSF, GM-CSF and MCP-1 in mesenteric lymph nodes were significantly decreased in mice compared to those in the Vac group, while concentrations of IL-1α, IL-2, IL-9, IL-13,G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1α and TNF-α were significantly increased in serum compared to the Vac group. In conclusion, the results presented here indicate that exposure to DON at 2.0 mg/L via drinking water can disrupt the immune response in vaccinated mice by modulating cytokines and chemokines involved in their immune response to infectious disease.     

Flavobacterium kyungheensis sp. nov., isolated from soil of a ginseng field

A Gram-staining negative, strictly aerobic, motile by gliding, non-spore-forming, pale yellow pigmented and rod-shaped bacterium designated strain THG-107^T was isolated from soil of a ginseng field on Ganghwa Island in the Republic of Korea and its taxonomic position was investigated by using a polyphasic study. Growth of strain THG-107^T was found to occur at 4–37 °C (optimum, 20–30 °C), at pH 5.5–10 (optimum, pH 7.0) and in the presence of 0–1 % (w/v) NaCl (optimum, absence) on R2A agar. On the basis of 16S rRNA gene sequence similarity, strain THG-107^T was shown to belong to the family Flavobacteriaceae and was related to Flavobacterium denitrificans ED5^T (99.1 % similarity). The G+C content of the genomic DNA was determined to be 34.2 mol%. These results are consistent with characteristics of members of the genus Flavobacterium. The only isoprenoid quinone detected in strain THG-107^T was menaquinone-6 (MK-6) and the major polyamine was identified as homospermidine (82.9 %). The major polar lipid detected was phosphatidylethanolamine and the major cellular fatty acids were identified as iso-C_15:0 (26.3 %), iso-C_17:0 3OH (12.6 %) and summed feature 3 (comprising C_16:1 ω7c and/or C_16:1 ω6c; 11.6 %). Flexirubin-type pigments were found to be present. Strain THG-107^T has β-glucosidase activity to convert ginsenosides Rb₁ and Rd into Gyp17 and F₂. DNA-DNA hybridization with F. denitrificans ED5^T was 52 %. Strain THG-107^T could be distinguished from F. denitrificans ED5^T and the other species of the genus Flavobacterium by its phylogenetic and genetic distinctiveness and by several phenotypic properties. Therefore, strain THG-107^T is considered to represent a novel species in the genus Flavobacterium, for which the name Flavobacterium kyungheensis sp. nov. is proposed (type strain THG-107^T = KACC 16219^T = LMG 26575^T).     

FAM111A Mutations Result in Hypoparathyroidism and Impaired Skeletal Development

Kenny-Caffey syndrome (KCS) and the similar but more severe osteocraniostenosis (OCS) are genetic conditions characterized by impaired skeletal development with small and dense bones, short stature, and primary hypoparathyroidism with hypocalcemia. We studied five individuals with KCS and five with OCS and found that all of them had heterozygous mutations in FAM111A. One mutation was identified in four unrelated individuals with KCS, and another one was identified in two unrelated individuals with OCS; all occurred de novo. Thus, OCS and KCS are allelic disorders of different severity. FAM111A codes for a 611 amino acid protein with homology to trypsin-like peptidases. Although FAM111A has been found to bind to the large T-antigen of SV40 and restrict viral replication, its native function is unknown. Molecular modeling of FAM111A shows that residues affected by KCS and OCS mutations do not map close to the active site but are clustered on a segment of the protein and are at, or close to, its outer surface, suggesting that the pathogenesis involves the interaction with as yet unidentified partner proteins rather than impaired catalysis. FAM111A appears to be crucial to a pathway that governs parathyroid hormone production, calcium homeostasis, and skeletal development and growth.     

Oxysterols induce transition of monocytic cells to phenotypically mature dendritic cell-like cells

Dendritic cells (DCs) activate adaptive immune responses in atherosclerotic plaques; however, the origin of DCs is in question. We attempted to determine whether cholesterol or its oxide forms, which are detected in abundance in atheromatous lesions, could induce differentiation or transition of monocytic cells to DCs. Treatment of THP-1 cells with 27-hydroxycholesterol (27OH-Chol) and 7α-hydroxycholesterol (7αOH-Chol) resulted in an increase in the numbers of adherent cells, and, in contrast to PMA, decreased uptake of FITC-conjugated dextran. In addition, treatment with 27OH-Chol and 7αOH-Chol induced expression of mDC-specific molecules, including CD40, CD80, CD83, and CD88. Of the two oxysterols, 27OH-Chol enhanced expression of MHC class I and II molecules as well as CCR7. However, treatment with an identical concentration of cholesterol and 7-ketocholesterol did not influence adherence, uptake of FITC-conjugated dextran, and expression of the aforementioned molecules. This is the first study to report on change of monocytic cells by oxysterols to phenotypically atypical cells with some characteristics of mDCs detected in atherosclerotic lesions. We propose that a certain type of oxysterol would contribute to immune responses in atherosclerotic lesions by enhancing expression of multiple CD molecules as well as MHC molecules by monocytic cells.