• Korean Journal of Plant Tissue Culture
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• v.24 no.4
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• pp.197-201
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• 1997

Plant seed oil-bodies or oleosomes ate the repository of the neutral lipid stored in seeds. These organelles in many oilseeds may comprise half of the total cellular volume. Oleosomes are surrounded by a half-unit membrane of phospholipid into which are embedded proteins called oleosins. Oleosins are present at high density on the oil-body surface and after storage proteins comprise the most abundant proteins in oilseeds. Oleosins are specifically targeted and anchored to oil-bodies after co-translation on the ER. It has been shown that the amino-acid sequences responsible for this unique targeting reside primarily in the central hydrophobic tore of the oleosin polypeptide. In addition, a signal-like sequence is found near the junction of the hydrophobic domain and ann N-terminal hydrophilic / amphipathic domain. This "signal" which is uncleaved is also essential for correct targeting. Oil-bodies and their associated oleosins may be recovered by floatation centrifugation of aqueous seed extracts. This simple partitioning step results in a dramatic enrichment for oleosins in the oil-body fraction. In the light of these properties, we reasoned that it would be feasible to create fusion proteins on oil-bodies comprising oleosins and an additional valuable protein of pharmaceutical or industrial interest. It was further postulated that if these proteins were displayed on the outer surface of oil-bodies, it would be possible to release them from the purified oil-bodies using chemical or proteolytic cleavage. This could result in a simple means of recovering high-value protein from seeds at a significant (i.e. commercial) scale. This procedure has been successfully reduced to practice for a wide variety of proteins of therapeutic, industrial and food no. The utillity of the method will be discussed using a blood anticoagulant, hirudin, and industrial enzymes as key examples.

• Molecules and Cells
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• v.38 no.5
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• pp.402-408
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• 2015

Protein O-GlcNAcylation, dictated by cellular UDP-N-acetylglucosamine (UDP-GlcNAc) levels, plays a crucial role in posttranslational modifications. The enzyme GlcNAc kinase (NAGK, E.C. 2.7.1.59) catalyzes the formation of GlcNAc-6-phosphate, which is a major substrate for the biosynthesis of UDP-GlcNAc. Recent studies have revealed the expression of NAGK in different types of cells especially in neuronal dendrites. Here, by immunocytochemistry (ICC) and immunonucleochemistry (INC) of cultured rat hippocampal neurons, HEK293T and GT1-7 cells, we have showed that NAGK immuno-reactive punctae being present in the nucleoplasm colocalized with small nuclear ribonucleoprotein-associated protein N (snRNPN) and p54NRB, which are speckle and paraspeckle markers, respectively. Furthermore, NAGK IR cluster was also found to be colocalized with GTF2H5 (general transcription factor IIH, polypeptide 5) immuno reactive punctae. In addition, relative localization to the ring of nuclear lamin matrix and to GlcNAc, which is highly enriched in nuclear pore complexes, showed that NAGK surrounds the nucleus at the cytoplasmic face of the nuclear outer membrane. By in situ proximity ligation assay (PLA) we confirmed the colocalization of NAGK with snRNPN in the nucleus and in dendrites, while we also verified the interactions of NAGK with p54NRB, and with GTF2H5 in the nucleus. These associations between NAGK with speckle, paraspeckle and general transcription factor suggest its regulatory roles in gene expression.

• Journal of Applied Biological Chemistry
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• v.58 no.3
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• pp.227-232
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• 2015

A microarray study has been employed to understand changes of gene expression in E. coli KD43162 resistant to ampicillin, ampicillin-sulbactam, piperacillin, piperacillin-tazobactam, cefazolin, cefepime, aztreonam, imipenem, meropenem, gentamicin, tobramycin, ciprofloxacin, levofloxacin, moxifloxacin, fosfomycin, and trimethoprim-sulfamethoxazole except for amikacin using disk diffusion assay. Using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and MALDI-TOF MS analyses, 36 kDa of outer membrane proteins (OMPs) was found to be deleted in the multidrug resistant E. coli KD 43162. Microarray analysis was used to determine up- and down-regulated genes in relation to multidrug resistant E. coli KD43162. Among the up-regulated genes, these genes were corresponded to express the proteins as penicillin-binding proteins (PBPs), tartronate semialdehyde reductase, ethanolamine utilization protein, shikimate kinase I, allantoinase, predicted SAM-dependent methyltransferase, L-glutamine: D-fructose-6-phosphate aminotransferase (GFAT), phospho-glucosamine mutase, predicted N-acetylmannosamine kinase, and predicted N-acetylmannosamine-6-P epimerase. Up-regulation of PBPs, one of primary target sites of antibiotics, might be responsible for the multidrug resistance in E. coli with increasing amount of target sites. Up-regulation of GFAT enzyme may be related to the up-regulation of PBPs because GFAT produces N-acetylglucosamine, a precursor of peptidoglycans. One of GFAT inhibitors, azaserine, showed a potent inhibition on the growth of E. coli KD43162. In conclusion, up-regulation of PBPs and GFATs with the loss of 36 kDa OMP refers the multidrug resistance in E. coli KD 43162.

• The Korean Journal of Pain
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• v.28 no.1
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• pp.4-10
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• 2015

Etifoxine (etafenoxine, $Stresam^{(R)}$) is a non-benzodiazepine anxiolytic with an anticonvulsant effect. It was developed in the 1960s for anxiety disorders and is currently being studied for its ability to promote peripheral nerve healing and to treat chemotherapy-induced pain. In addition to being mediated by $GABA_A{\alpha}2$ receptors like benzodiazepines, etifoxine appears to produce anxiolytic effects directly by binding to ${\beta}2$ or ${\beta}3$ subunits of the $GABA_A$ receptor complex. It also modulates $GABA_A$ receptors indirectly via stimulation of neurosteroid production after etifoxine binds to the 18 kDa translocator protein (TSPO) of the outer mitochondrial membrane in the central and peripheral nervous systems, previously known as the peripheral benzodiazepine receptor (PBR). Therefore, the effects of etifoxine are not completely reversed by the benzodiazepine antagonist flumazenil. Etifoxine is used for various emotional and bodily reactions followed by anxiety. It is contraindicated in situations such as shock, severely impaired liver or kidney function, and severe respiratory failure. The average dosage is 150 mg per day for no more than 12 weeks. The most common adverse effect is drowsiness at the initial stage. It does not usually cause any withdrawal syndromes. In conclusion, etifoxine shows less adverse effects of anterograde amnesia, sedation, impaired psychomotor performance, and withdrawal syndromes than those of benzodiazepines. It potentiates $GABA_A$ receptor-function by a direct allosteric effect and by an indirect mechanism involving the activation of TSPO. It seems promising that non-benzodiazepine anxiolytics including etifoxine will replenish shortcomings of benzodiazepines and selective serotonin reuptake inhibitors according to animated studies related to TSPO.

• Restorative Dentistry and Endodontics
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• v.40 no.4
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• pp.306-313
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• 2015

Objectives: Molecular mechanism of the pathogenicity of Enterococcus faecalis (E. faecalis), a suspected endodontic pathogen, has not yet been adequately elucidated due to limited information on its virulence factors. Here we report the identification of in vivo expressed antigens of E. faecalis by using a novel immunoscreening technique called change-mediated antigen technology (CMAT) and an experimental animal model of endodontic infection. Materials and Methods: Among 4,500 E. coli recombinant clones screened, 19 positive clones reacted reproducibly with hyperimmune sera obtained from rabbits immunized with E. faecalis cells isolated from an experimental endodontic infection. DNA sequences from 16 of these in vivo-induced (IVI) genes were determined. Results: Identified protein antigens of E. faecalis included enzymes involved in housekeeping functions, copper resistance protein, putative outer membrane proteins, and proteins of unknown function. Conclusions: In vivo expressed antigens of E. faecalis could be identified by using a novel immune-screening technique CMAT and an experimental animal model of endodontic infection. Detailed analysis of these IVI genes will lead to a better understanding of the molecular mechanisms involved in the endodontic infection of E. faecalis.

• Journal of Microbiology and Biotechnology
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• v.25 no.4
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• pp.526-536
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• 2015

Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira, a genus of which more than 250 serovars have been identified. Commercial bacterin vaccines are limited in that they lack both cross-protection against heterologous serovars and long-term protection. This study investigated in mice the immunogenicity of an anti-leptospirosis vaccine, using the outer membrane proteins LipL32 and Loa22 as antigens. The immunogenicity of this vaccine formulation was compared with those induced by vaccines based on LipL32 or Loa22 alone. A DNA-encapsulated chitosan nanoparticle was used for in vivo DNA delivery. Using a unique DNA plasmid expressing both lipL32 and loa22 for vaccination, higher antibody responses were induced than when combining plasmids harboring each gene separately. Therefore, this formulation was used to test the immunogenicity when administered by a heterologous prime (DNA)-boost (protein) immunization regimen. The specific antibody responses against LipL32 (total IgG and IgG1) and Loa22 (IgG1) were higher in mice receiving two antigens in combination than in those vaccinated with a single antigen alone. Although no significant difference in splenic CD4⁺ T cell proliferation was observed among all groups of vaccinated mice, splenocytes from mice vaccinated with two antigens exhibited higher interferon-γ and IL-2 production than when using single antigens alone upon in vitro restimulation. Taken together, the immunogenicity induced by LipL32 and Loa22 antigens in a heterologous primeboost immunization regimen using chitosan as a DNA delivery system induces higher immune response, and may be useful for developing a better vaccine for leptospirosis.

• Journal of Life Science
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• v.18 no.2
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• pp.241-248
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• 2008

This study has been carried out to secretion antibodies for the purpose of preventing the infection of Helicobacter pylori and using them as a supplement for treatment. This experiments have been separated antigens from H. pylori and observed into antibody production and the agglutination of H. pylori for the separated antigens. As major antigenic proteins separated from H. pylori, the following could be verified: 12 kinds of band for whole cell (WC), seven kinds of band for outer membrane protein (OMP), three kinds of band for crude urease, and one kind of band for lipopolysaccharide (LPS). The IgG anti-H. pylori antibody of separated antigens showed $77.9{\pm}6.4{\mu}g/ml$ for we (L), $84.9{\pm}6.4{\mu}g/ml$ for OMP, and $123.8{\pm}2.9{\mu}g/ml$ for crude urease, at the same antigen concentration of $20{\mu}g/100ull$, which showed the most at the crude urease. And it turned out that the IgA antibodies were generated with $2.5{\pm}0.32{\mu}g/ml$ for WC (L), $2.0{\pm}0.43{\mu}g/ml$ for OMP, and $1.3{\pm}0.25{\mu}g/ml$ for crude urease, which demonstrated the most for WC (L) antigens. As a result of verifying the immunogenecity of antigenic protein through the Western blotting, major antigenic substances could be confirmed as follows: 10 kinds for WC, six kinds for OMP and three kinds for crude urease. The agglutination values on the H. pylori of the antibody were $2^5,\;2^5,\;2^6\;and\;2^7$ at the antigen serums of anti-WC (H), anti-WC (L), anti-OMP and anti-crude urease, respectively, which indicated the highest for the antigen serum of anti-crude urease. The urease activation-inhibiting absorbance of antigen serum created by each antigen was $0.14{\pm}0.01$ for WC (H), $0.16{\pm}0.01$ for WC (L), $0.18{\pm}0.03$ for OMP, and $0.18{\pm}0.04$ for urease, demonstrating a significant inhibiting effect, compared with $0.26{\pm}0.02$ of the control group.

• Molecules and Cells
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• v.38 no.12
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• pp.1064-1070
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• 2015

Translocator protein 18 kDa (TSPO) is a mitochondrial outer membrane protein and is abundantly expressed in a variety of organ and tissues. To date, the functional role of TSPO on vascular endothelial cell activation has yet to be fully elucidated. In the present study, the phorbol 12-myristate 13-acetate (PMA, 250 nM), an activator of protein kinase C (PKC), was used to induce vascular endothelial activation. Adenoviral TSPO overexpression (10-100 MOI) inhibited PMA-induced vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) expression in a dose dependent manner. PMA-induced VCAM-1 expressions were inhibited by Mito-TEMPO ($0.1-0.5{\mu}m$), a specific mitochondrial antioxidants, and cyclosporin A ($1-5{\mu}m$), a mitochondrial permeability transition pore inhibitor, implying on an important role of mitochondrial reactive oxygen species (ROS) on the endothelial activation. Moreover, adenoviral TSPO overexpression inhibited mitochondrial ROS production and manganese superoxide dismutase expression. On contrasts, gene silencing of TSPO with siRNA increased PMA-induced VCAM-1 expression and mitochondrial ROS production. Midazolam ($1-50{\mu}m$), TSPO ligands, inhibited PMA-induced VCAM-1 and mitochondrial ROS production in endothelial cells. These results suggest that mitochondrial TSPO can inhibit PMA-induced endothelial inflammation via suppression of VCAM-1 and mitochondrial ROS production in endothelial cells.

• The Korean Journal of Zoology
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• v.11 no.1
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• pp.5-12
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• 1968

The membrane structures were electron microscopically studied in the elongating lens fibers of the regenerating lens of adult Triturus pyrrhogaster. Observations were focused on the endoplasmic reticulum and mitochondria. The endoplasmic reticulum developed in the vicinity of the nucleus with active blebbing of the outer membrane. At the same time, the concentration of mitochondria around the rough-surfaced endoplasmic reticulum near by the nucleus was always observed. Both endoplasmic reticulum and mitochondrion undergo disintegration in the apical portion apart from the nucleus. Some considerations were discussed with reference to published data.

• Journal of Animal Science and Technology
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• v.46 no.3
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• pp.495-502
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• 2004

Intimin, the product of eae gene in EHEC O157:H7, is required for intimate adherence. In this study, the C-terminaI region(281 amino acids) of the EHEC OI57:H7 intimin were expressed as a protein fusion with (His)$_6$ which was used to raise antiserum in rabbits. The antiserum reacted in western blot with a 94kDa outer membrane protein of EHEC O157:H7. It was observed that the antibody titers both in egg yolk and serum appeared in 2${\sim}$4 weeks after immunization with fusion protein. At the time of 8 weeks, the titre of egg yolk was found to be higher than that of sera. According to the results of neutralization test, chicken egg-yolk antibody(lgY) against the recombinant intimin strongly reacted to EHEC O157:H7. We conclude that a truncated recombinant intimin could be used as an immunogen to elicit antibody(lgY) against O157:H7.