• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.278-281
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• 2004

The $\alpha$1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 plays a key role for the outer chain initiation of the N-linked oligosaccharides. A search for Hansenula polymorpha genes homologous to S. cerevisiae OCHI (ScOCH1) has revealed seven open reading frames (ORF100, ORF142, ORF168, ORF288, ORF379, ORF576, ORF580). All of the seven ORFs are predicted to be a type II integral membrane protein containing a transmembrane domain near the amino-terminal region and has a DXD motif, which has been found in the active site of many glycosyltransferases. Among this seven-membered OCH1 gene family of H. polymorpha, we have carried out a functional analysis of H. polymorpha ORF168 (HpOCH2) showing the highest identity to ScOCH1. Inactivation of this protein by disruption of corresponding gene resulted in several phenotypes suggestive of cell wall defects, including hypersensitivity to hygromycin B and sodium deoxycholate. The structural analysis of N-glycans synthesized in HpOCH2-disrupted strain (Hpoch2Δ) and the in vitro $\alpha$1,6-mannosyltransferase activity assay strongly indicate that HpOch2p is a key enzyme adding the first $\alpha$1,6-mannose residue on the core glycan Man$_{8}$GlcNAc$_2$. The Hpoch2Δ was further genetically engineered to synthesize a recombinant glycoprotein with the human compatible N-linked oligosaccharide, Man$_{5}$GlcNAc$_2$, by overexpression of the Aspergillus saitoi $\alpha$1,2-mannosidase with the 'HDEL” ER retention signal.gnal.

• Journal of Microbiology and Biotechnology
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• v.15 no.6
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• pp.1214-1220
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• 2005

Decreased porin-mediated outer membrane penetration of hydrophilic antibiotics is a common mechanism of antibiotic resistance in Gram-negative bacteria. This study was undertaken to determine whether a null mutation in Pseudomonas putida would suppress porin synthesis, and therefore reduce the susceptibility of the organism to streptomycin, norfloxacin, and tetracycline. Inverse PCR amplification and double-stranded DNA sequencing were used to identify chromosomal genes carrying TnphoA'-1 inserts. Genome database available was used to identify putative homologue genes, one of which encodes protein with homology to domains of the MutS of P. putida, suggesting a crucial role in the multidrug resistance. Increased resistance to streptomycin, norfloxacin, and tetracycline might be due to accumulation of compensatory mutations. Either no growth or slow growth was observed in P. putida KH1027 when grown in minimal medium containing gluconate, glucose, or citrate; however, it is not clear whether the growth patterns contributed to the multidrug resistance.

• The Journal of the Korean Society for Microbiology
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• v.35 no.4
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• pp.309-316
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• 2000

This study was focused on the isolation of pathogenic Vibrio species, V. vulnificus and V. parahaemolyticus from marine environment from May to July of 1999. Isolation sites were coast near by Pusan and Daechon. The results obtained were as follows: 1. Seventy strains of V. parahaemolyticus and 19 strains of V. vulnificus were isolated from a total of 120 specimens. 2. Nineteen strains of V. vulnificus did not fermented arabinose and salicin but fermented lactose and cellobiose. All of V. parahaemolyticus isolates did not fermented lactose and cellobiose. 47 strains of V. parahaemolyticus fermented arabinose but 53 strains did not fermented salicin. 3. V. vulnificus and V. parahaemolyticus isolates showed three different API index numbers with 5046105 and 4346107 dominant. 4. V. vulnificus did not grow on 0% and 8% NaCl containing medium. V. parahaemolyticus grew on 8% NaCl containing medium. 5. V. vulnificus isolates and V. parahaemolyticus revealed different outer membrane protein profiles on SDS-PAGE.

• 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.

• 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.

• 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.

• 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.

• 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.

• Korean Journal of Veterinary Service
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• v.33 no.3
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• pp.223-231
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• 2010

A total of 191 samples collected from the commercial swine farms located in Chungnam province were investigated by PCR to estimate the prevalence of Lawsonia (L.) intracellularis infection. In the group of the pigs with proliferative enteritis, 14 (93.3%) of 15 intestinal samples and 12 (80.0%) of 15 feces were positive in PCR. In contrast, a relatively low positive rate (18.0%, 29 of 161 samples) was determined in the group of normal healthy pigs. The group of pigs over 120 days showed the highest positive rates (26.8%, 15 of 56 samples). In the comparison of the sequences of 210bp for species specific fragments and 301bp for outer membrane protein, the isolates (L1. L2) showed almost 100% identity with the reference L. intracellularis (L08049, USA). For the sequences of partial 16s rDNA, the homologies among the 5 isolates (L1-L5) were 97.4% to 99.3%, and those of 5 sequences (L1-L5) versus 5 overseas reference strains of L. intracellularis ranged from 98.6% to 99.8%. In the comparison of the nucleotide sequences among 5 isolates and other species in Desulfovibrionales showed 82.4 to 99.5% identities. The 5 isolates shared relatively low identities (76.9% to 84.4%) with the species of alpha-proteobacteria. In phylogenetic analysis based on the 16s rDNA sequences, all of the 5 isolates (L1-L5) were located in the same branch with the strains of L. intracellularis that were previously isolated from the pigs in USA and China. Seven strains of Desulfovibrio sp. were clustered in the neighboring branches, whereas alpha and gamma Proteobacteria showed distant relationship with L. intracellularis strains. The present findings suggest that L. intracellularis infection is endemic in the swine farms in the regions, and that the domestic isolates maintained very limited genetic variation.

• 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.