A nonfusogenic antigen mimic of influenza hemagglutinin glycoproteins constituted with soluble full-length HA1 and truncated HA2 proteins expressed in E. coli.

A novel method is proposed to produce a soluble recombinant antigen mimic, constituted with full-length HA1 and truncated HA2 individually expressed in E. coli, instead of a precursor form of hemagglutinin protein, that is similar to the naturally processed and disulfide-linked HA1/HA2 on the envelope of the influenza A virus strain X-31 (H3N2). A truncated ectodomain of HA2 subunit, HA2(23-185)/C137S, lacked two membrane-interacting sequences, i.e., the N-terminal fusion peptide as well as the transmembrane domain and short cytoplasmic segment at the C terminus. A recombinant HA1 (rHA1) subunit protein, HA1(1-328)/C14S/L157S, lacked the signal peptide. Mutations C137S and C14S in the HA2 and HA1 subunits, respectively, were introduced to prevent any possible disulfide linkage between the two subunit proteins. The rHA antigen mimic would be nonfusogenic mainly due to the absence of the N-terminal fusion peptide as well as the C-terminal transmembrane domain in the truncated HA2, and eventually less cytotoxic as well. Antibody responses induced by two soluble rHA antigens were evaluated by ELISA assays to detect rHA antigens injected and to validate both anti-HA1 and anti-HA2 antibodies produced in the mice sera. Antigenic rHA proteins also elicited neutralizing antibodies against homologous H3N2 influenza virus in the immunized mice, without severe body weight loss or any other adverse symptoms.

Acid hydrolysis of Curcuma longa residue for ethanol and lactic acid fermentation.

This research examines the acid hydrolysis of Curcuma longa waste, to obtain the hydrolysate containing lactic acid and ethanol fermentative sugars. A central composite design for describing regression equations of variables was used. The selected optimum condition was 4.91% sulphuric acid, 122.68°C and 50 min using the desirability function under the following conditions: the maximum reducing sugar (RS) yield is within the limited range of the 5-hydroxymethylfurfural (HMF) and furfural concentrations. Under the condition, the obtained solution contained 144 g RS/L, 0.79 g furfural/L and 2.59 g HMF/L and was directly fermented without a detoxification step. The maximum product concentration, average productivity, RS conversion and product yield were 115.36 g/L, 2.88 g/L/h, 89.43% and 64% for L-lactic acid; 113.92 g/L, 2.59 g/L/h, 88.31% and 63.29% for D-lactic acid; and 55.03 g/L, 1.38 g/L/h, 42.66 and 30.57%, respectively, for ethanol using a 7-L jar fermenter.

PTH regulates myleoid ELF-1-like factor (MEF)-induced MAB-21-like-1 (MAB21L1) expression through the JNK1 pathway.

Continuous treatment with parathyroid hormone (PTH) or excess endogenous PTH due to primary hyperparathyroidism causes increased bone resorption and, subsequently, decreased bone volume. Our previous studies showed that myeloid Elf-1-like factor (MEF) not only suppresses osteoblast differentiation through inhibition of Runx2 activity and other osteogenesis-related genes but also specifically increases the expression of Mab21, a potential transcriptional repressor of osteoblast differentiation. Here we show that the JNK1 pathway is involved in the MEF-mediated up-regulation of Mab21 expression due to PTH stimulation. PTH increased the transcription level of Mab21 in MG63 human osteoblastic cells, in contrast to the suppressive effect of TGFß1. PTH phosphorylates serine residues of MEF as well as c-Jun, a known substrate of JNK1. By in vitro kinase assay, we confirmed that MEF is phosphorylated by JNK1, but not by ERK. Co-transfection of MEF with both MKK4 and JNK1 increased the promoter activity of Mab21 in CV1 cells significantly more than MEF alone. We also identified the phosphorylation of MEF serine 641 by in vitro and in vivo JNK1 kinase assays combined with a proteomics approach. In conclusion, our findings indicate that MEF is involved in PTH suppression of osteoblasts through activating the MKK4/JNK1 pathway and subsequently up-regulating Mab21 expression.

Pusillimonas harenae sp. nov., isolated from a sandy beach, and emended description of the genus Pusillimonas.

A Gram-stain-negative, motile bacterium with two lateral flagella, designated strain B201(T), was isolated from beach sand from the Taean coast in South Korea. Cells were ovoid rods and positive for catalase and oxidase. Growth of strain B201(T) was observed between 15 and 45 °C (optimum, 30 °C) and between pH 5.0 and 9.0 (optimum, pH 6.0-7.5). Strain B201(T) contained ubiquinone Q-8 as the major isoprenoid quinone, but MK-6 was also present as a minor quinone. The major fatty acids of strain B201(T) were C(17:0) cyclo, C(16:0), summed feature 2 (iso-C(16:1) I/C(14:0) 3-OH and/or C(12:0) ALDE), C(12:0) and C(19:0) cyclo ω8c. The major cellular polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid and three aminolipids. The genomic DNA G+C content was 53.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain formed a phyletic lineage with Pusillimonas ginsengisoli DCY25(T) within the genus Pusillimonas. Strain B201(T) was most closely related to P. ginsengisoli DCY25(T) and Pusillimonas soli MJ07(T) with similarities of 98.6 and 97.5%, respectively. However, DNA-DNA relatedness values of strain B201(T) with P. ginsengisoli DCY25(T) and P. soli MJ07(T) were 30.2±5.4 and 4.9±1.8%, respectively. On the basis of chemotaxonomic data and molecular properties, strain B201(T) represents a novel species of the genus Pusillimonas, for which the name Pusillimonas harenae sp. nov. is proposed; the type strain is B201(T) (=KACC 14927(T)=JCM 16917(T)). An emended description of the genus Pusillimonas is given.

Luteimonas lutimaris sp. nov., isolated from a tidal flat.

A Gram-staining-negative, strictly aerobic bacterium, designated strain G3(T), was isolated from a tidal flat of the Taean coast in South Korea. Cells were moderately halotolerant and non-motile rods showing catalase- and oxidase-positive reactions. Growth of strain G3(T) was observed between 15 and 40 °C (optimum 30 °C) and between pH 5.5 and 9.0 (optimum pH 6.5-7.5). Strain G3(T) contained Q-8 as the predominant lipoquinone and iso-C(15 : 0), iso-C(17 : 1)ω9c, iso-C(16 : 0) and iso-C(11 : 0) as the major fatty acids. The G+C content of the genomic DNA was 69.6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain G3(T) formed a tight phylogenetic lineage with Luteimonas mephitis B1953/27.1(T) within the genus Luteimonas and was most closely related to L. mephitis B1953/27.1(T) with 98.0 % 16S rRNA gene sequence similarity. The DNA-DNA relatedness between strain G3(T) and L. mephitis B1953/27.1(T) was 35.2 ± 3.3 %. On the basis of chemotaxonomic data and molecular properties, strain G3(T) represents a novel species of the genus Luteimonas, for which the name Luteimonas lutimaris sp. nov. is proposed. The type strain is G3(T) (= KACC 14929(T) = JCM 16916(T)).

Hypoxanthine levels in human urine serve as a screening indicator for the plasma total cholesterol and low-density lipoprotein modulation activities of fermented red pepper paste.

Fermented red pepper paste (FRPP) is one of the most well-known traditional foods in Korea. The effects of FRPP in experimental animals and adipocytes have been previously reported. However, the biochemical effects have not yet been validated in humans with various genetic backgrounds and environmental factors. In this study, 28 female volunteers (body mass index, more than 23 kg/m(2)) aged 19 to 60 years were treated with either FRPP or a placebo for 12 weeks. Marked cholesterol modulation was observed in the FRPP-treated group compared with the placebo group. Although the baseline (pretreatment) total cholesterol and low-density lipoprotein levels and body mass index of the volunteers did not differ significantly between the placebo- and FRPP-treated groups, FRPP caused a modulation of cholesterol levels not seen in the placebo group, causing either no variation or a decrease in low-density lipoprotein and total cholesterol levels. Thus, urinary metabolomic profiling of pretreatment samples was carried out in these 2 FRPP-treated groups using (1)H-nuclear magnetic resonance-based metabolomic techniques. These 2 groups, with their opposing cholesterol-modulation tendencies, could be clearly differentiated by orthogonal projections to latent structures-discriminant analysis-derived score plots. In addition, their levels of hypoxanthine differed markedly. We propose that urinary hypoxanthine levels can be used as a screening biomarker to predict the efficacy of the cholesterol-modulating activity of FRPP in human subjects.

Strain development of Streptomyces sp. for tacrolimus production using sequential adaptation.

An immunosuppressant tacrolimus-producing strain of Streptomyces sp. TST8 was isolated and developed by the TS corporation in Korea using the sequential adaptation of media containing tacrolimus (600-1600 mg/l). The aim of the tacrolimus sequential adaptation protocol was to select those cells with tacrolimus resistance and to reduce product inhibition of the tacrolimus-producing strain. The developed strains produced more tacrolimus than the original strain. In particular, the TST10 strain adapted in the medium containing 900 mg/l of tacrolimus produced 972 mg/l of tacrolimus in the final titer after 7 days of cultivation in a 5-l jar fermenter. This is the largest final titer of tacrolimus produced by a specific strain to date. Because the sequential adaptation protocol is limited by the solubility of tacrolimus in water, the final tacrolimus titer of TST11 adapted in the medium containing 1600 mg/l of tacrolimus was lower than that of TST10. The developed strains and the development method using sequential adaptation can facilitate the efficient and economical production of tacrolimus.