• Microbiology and Biotechnology Letters
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• v.44 no.3
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• pp.355-362
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• 2016

The phosphomannomutase/phosphoglucomutase gene (pmm/pgm) of Vibrio anguillarum (the causative agent of fish vibriosis) was cloned, and the open reading frame corresponded to a protein with 446 amino acids. The pmm/pgm gene showed a significant degree of sequence homology with the previously reported genes from V. mimicus, V. vulnificus, V. splendidus, and V. harveyi, with 92.3%, 91.4%, 89.9%, and 89.9% amino acid identity, respectively. By reverse transcriptase-polymerase chain reaction, we found that the pmm/pgm gene was upregulated under cold stress condition. The PMM/PGM protein is known to catalyze the interconversion between mannose-1-phosphate and mannose-6-phosphate or glucose-1-phosphate and glucose-6-phosphate, which are important intermediates for lipopolysaccharide (LPS) biosynthesis. To confirm the role of PMM/PGM in the LPS biosynthetic pathway, we constructed a knock out mutant by homologous recombination. The respective LPSs were isolated from the V. anguillarum wild-type and mutant strains, and changes were compared by subjecting them to sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on the different patterns of the LPSs, we expect the pmm/pgm gene to have an important role in LPS biosynthesis. The pmm/pgm-deficient mutant of V. anguillarum will contribute to further studies about the role of LPS in V. anguillarum pathogenesis.

• Journal of the Korean Society of Food Science and Nutrition
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• v.34 no.5
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• pp.573-580
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• 2005

Cholesterol $7\alpha-hydroxylase$ (CYP7A1) is the rate-limiting enzyme in the conversion of cholesterol to bile acids and plays a central role in regulating cholesterol homeostasis. We previously showed that a fermentable $\beta-glucan$ ingestion decreased plasma cholesterol levels due to fecal bile acid excretion elevation involved inincrease of cholesterol $7\alpha-hydroxylase$ mRNA expression and activity. It is proposed that short chain fatty acids (SCFA) produced by cecal and colonic fermentation of soluble fiber are associated with cholesterol-lowering effect of fiber. In the present study, we investigated whether CYP7A1 expression is up-regulated by short chain fatty acids or by hormones in cultured human hepatoma (HepG2) cells. Confluent HepG2 cell were incubated with acetate, propionate, or butyrate at 1 mM concentration for 24 hrs. Acetate as well as propionate increased to 1.8-fold expression of CYP7A1 mRNA than the control. Butyrate also increased 1.5-fold expression of CYP7A1 mRNA. Our data show for the first time that SCFA increase expression of CYP7A1 mRNA. Adding insulin, dexamethasone and triiodothyronine $(1\;{\mu}M)$ to HepG2 cell increased the expression of CYP7A1 mRNA to $150\%,\;173\%,\;141\%$, respectively. These results suggest that SCFA produced by cecal fermentation stimulate enteric nervous system, in which secreted some neuropeptides may be responsible for change in cholesterol and bile acid metabolism. These findings suggest that SCFA are involved in lowering plasma cholesterol levels due to the up-regulation of CYP7A1 and bile acid synthesis.

• Nuclear Medicine and Molecular Imaging
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• v.41 no.3
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• pp.241-246
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• 2007

Purpose: $[^{11}C]6-OH-BTA-1$ ([N-methyl-$^{11}C$]2-(4'-methylaminophenyl)-6-hydroxybenzothiazole, 1), a -amyloid imaging agent for the diagnosis of Alzheimer's disease in PET, can be labeled with higher yield by a simple loop method. During the synthesis of $[^{11}C]1$, we found the formation of by-products in various solvents, e.g., methylethylketone (MEK), cyclohexanone (CHO), diethylketone (DEK), and dimethylformamide (DMF). Materials and Methods: In Automated radiosynthesis module, 1 mg of 4-aminophenyl-6-hydroxybenzothiazole (4) in 100 l of each solvent was reacted with $[^{11}C]methyl$ triflate in HPLC loop at room temperature (RT). The reaction mixture was separated by semi-preparative HPLC. Aliquots eluted at 14.4, 16.3 and 17.6 min were collected and analyzed by analytical HPLC and LC/MS spectrometer. Results: The labeling efficiencies of $[^{11}C]1$ were $86.0{\pm}5.5%$, $59.7{\pm}2.4%$, $29.9{\pm}1.8%$, and $7.6{\pm}0.5%$ in MEK, CHO, DEK and DMF, respectively. The LC/MS spectra of three products eluted at 14.4, 16.3 and 17.6 mins showed m/z peaks at 257.3 (M+1), 257.3 (M+1) and 271.3 (M+1), respectively, indicating their structures as 1, 2-(4'-aminophenyl)-6-methoxybenzothiazole (2) and by-product (3), respectively. Ratios of labeling efficiencies for the three products $([^{11}C]1:[^{11}C]2:[^{11}C]3)$ were $86.0{\pm}5.5%:5.0{\pm}3.4%:1.5{\pm}1.3%$ in MEK, $59.7{\pm}2.4%:4.7{\pm}3.2%:1.3{\pm}0.5%$ in CHO, $9.9{\pm}1.8%:2.0{\pm}0.7%:0.3{\pm}0.1%$ in DEK and $7.6{\pm}0.5%:0.0%:0.0%$ in DMF, respectively. Conclusion: The labeling efficiency of $[^{11}C]1$ was the highest when MEK was used as a reaction solvent. As results of mass spectrometry, 1 and 2 were conformed. 3 was presumed.

• Tuberculosis and Respiratory Diseases
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• v.58 no.6
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• pp.590-599
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• 2005

Object : Cigarette smoking is a major cause of mucus hypersecretion, which is a pathophysiological feature of many inflammatory airway diseases. Mucins, which are an important part of the airway mucus, are synthesized from the Muc gene in airway epithelial cells. However, the signaling pathways for cigarette smoke-induced mucin synthesis are unknown. The aim of this study was to determine the signal pathway for smoking induced Muc5ac gene expression. Methods : A549 cells were cultured and transiently transfected with the Muc5ac promoter fragment. These cells were stimulated with 5% cigarette smoke extract (CSE) alone or with CSE after a pretreatment with various signal transduction pathway inhibitors (AG1478, PD98059 and SB203580). The Muc5ac promoter activity was examined using the luciferase reporter system, and the level of phosphorylated EGFR, ERK1/2, p38 MAPK and JNK were all examined using Western blot analysis. Muc5ac mRNA expression was also examined using reverse transcriptase polymerase chain reactions (RT-PCR). Results : 1. The peak level of luciferase activity of the Muc5ac promoter was observed at 5% concentration and after 3 hours of incubation with the CSE. The level of EGFR phosphorylation and the luciferase activity of the transfected cells caused by the CSE were significantly suppressed by AG1478 or PD98059 (P<0.01). 2. CSE phosphorylated ERK1/2 or p38 MAPK but not JNK. The Muc5ac mRNA expression level was increased by the CSE but that was suppressed by PD98059 or AG1478. 3. The CSE-induced phosphorylation of ERK1/2 was blocked by PD98059 and that of p38 MAPK was blocked by either PD98059 or SB203580. Either PD98059 or SB203580 suppressed the luciferase activity of the transfected cells (P<0.0001). Conclusion : The Muc5ac mRNA expression level was increased by the CSE. The increased CSE-induced transcriptional activity was mediated via EGF receptor activation, which led to ERK1/2 and p38 MAPK phosphorylation.