• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1343-1349
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• 2004

A native strain of Pasteurella multocida was isolated from pigs suffering from severe atrophic rhinitis at domestic farms in Gyeonggi Province, Korea, and was identified as capsular serogroup 'D' and somatic serotype '4' by disc diffusion decapsulation and gel diffusion precipitation tests, respectively. The P. multocida (D:4) induced atrophic rhinitis in healthy pigs by the secondary infection. The gene for outer membrane protein H (ompH) of P. multocida (D:4) was cloned in Escherichia coli DH5$\alpha$ by PCR. The open reading frame of the ompH was composed of 1,023 bp, possibly encoding a protein with 341 amino acid residues containing a signal peptide of 20 amino acids at N-terminus, and the gene product with molecular mass of ca. 38 kDa was identified by SDS-PAGE. Hydropathy profiles indicated that there are two variable domains in the OmpH. To express the ompH in E. coli, the gene was manipulated in various ways. Expression of the truncated as well as full-length forms of the recombinant OmpH was fatal to the host E. coli BL21 (DE3). However, the truncated OmpH fused with GST was consecutively expressed in E. coli DH5$\alpha$. A large quantity of the fused polypeptide was purified through GST-affinity chromatography.

• Journal of Microbiology and Biotechnology
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• v.12 no.2
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• pp.249-257
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• 2002

Pseudomonas fluorescens GL20 is a plant growth-promoting rhizobacterium that produces a large amount of hydroxamate siderophore under iron-limited conditions. The strain GL20 considerably inhibited the spore germination and hyphal growth of a plant pathogenic fungus, Fusarium solani, when iron was limited, significantly suppressed the root-rot disease on beans caused by F. solani, and enhanced the plant growth. The mechanism for the beneficial effect of strain GL20 on the disease suppression was due to the siderophore production, evidenced by mutant strains derived from the strain. Analysis of the outer membrane protein profile revealed that the growth of strain GL20 induced the synthesis of specific iron-regulated outer membrane proteins with molecular masses of 85- and 90 kDa as the high-affinity receptors for the ferric siderophore. In addition, a cross-feeding assay revealed the presence of multiple inducible receptors for heterologous siderophores in the strain. In order to induce increased efficacy and potential in biological control of plant disease, a siderophore-overproducing mutant, GL20-S207, was prepared by NTG mutagenesis. The mutant GL20-S207 produced nearly 2.3 times more siderophore than the parent strain. In pot trials of beans with F. solani, the mutant increased plant growth up to 1.5 times compared with that of the parent strain. These results suggest that the plant growth-promoting P. fluorescens GL20 and the genetically bred P. fluorescens GL20-S207 can play an important role in the biological control of soil-borne plant diseases in the rhizosphere.

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.555-568
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• 1997

Fowl typhoid caused by Salmonella gallinarum has increased dramatically since 1992 and has caused a great economic losses in chicken industry by characterizing with high mortality. In these studies, we investigated the immunogenicity and protectivity in chickens which were immunized with outer membrane protein(OMP) extracted from isolates of S gallinarum against challenge with live microorganism. Outer membrane proteins were composed of various sizes of molecular weight including 14K, 22K, 31K, 36K, 40K and 55K and the most of them responded strongly against rabbit antisera in immunoblot analysis. The chickens vaccinated with OMP or vaccinated with whole-cell combined with OMP($200{\mu}g$/chickens) complex showed higher delayed type hypersensitivity(DTH) response than that of whole-cell vaccinated group. The protective rates of OMP or whole-cell combined with OMP complex group against challenge of S gallinarum were higher (above 75%) than those (45~50%) of whole-cell vaccinated group. All vaccines were safe and the body weight-gains of all vaccinated groups were not significantly different (p<0.05) from those of nonvaccinated control group. In vitro tests, OMP stimulated both the proliferation of lymphocytes and T-lymphocytes, and OMP-induced lymphocyte proliferation was higher in the cells of the immunized chickens with OMP than in those from the control chickens.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.4
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• pp.255-264
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• 2012

The structures of the intact synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortexs, and the outer and the inner monolayer separately, were evaluated with 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1,3-di(1-pyrenyl)propane (Py-3-Py) as fluorescent reporters and trinitrophenyl groups as quenching agents. The methanol increased bulk rotational and lateral mobilities of SPMVs lipid bilayers. The methanol increased the rotational and lateral mobilities of the outer monolayers more than of the inner monolayers. n-(9-Anthroyloxy)stearic acid (n-AS) were used to evaluate the effect of the methanol on the rotational mobility at the 16, 12, 9, 6, and 2 position of aliphatic chains present in phospholipids of the SPMVs outer monolayers. The methanol decreased the anisotropy of the 16-(9-anthroyloxy)palmitic acid (16-AP), 12-(9-anthroyloxy)stearic acid (12-AS), 9-(9-anthroyloxy)stearic acid (9-AS), and 6-(9-anthroyloxy)stearic acid (6-AS) in the SPMVs outer monolayer but it increased the anisotropy of 2-(9-anthroyloxy)stearic acid (2-AS) in the monolayers. The magnitude of the increased rotational mobility by the methanol was in the order at the position of 16, 12, 9, and 6 of aliphatic chains in phospholipids of the outer monolayers. Furthermore, the methanol increased annular lipid fluidity and also caused membrane proteins to cluster. The important finding is that was far greater increase by methanol in annular lipid fluidity than increase in lateral and rotational mobilities by the methanol. Methanol alters the stereo or dynamics of the proteins in the lipid bilayers by combining with lipids, especially with the annular lipids. In conclusion, the present data suggest that methanol, in additions to its direct interaction with proteins, concurrently interacts with membrane lipids, fluidizing the membrane, and thus inducing conformational changes of proteins known to be intimately associated with membranes lipids.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.6
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• pp.413-422
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• 2012

The purpose of this study is to investigated the mechanism of pharmacological action of local anesthetic and provide the basic information about the development of new effective local anesthetics. Fluorescent probe techniques were used to evaluate the effect of lidocaine HCl on the physical properties (transbilayer asymmetric lateral and rotational mobility, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex, and liposomes of total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. An experimental procedure was used based on selective quenching of 1,3-di(1-pyrenyl)propane (Py-3-Py) and 1,6-diphenyl-1,3,5-hexatriene (DPH) by trinitrophenyl groups, and radiationless energy transfer from the tryptophans of membrane proteins to Py-3-Py. Lidocaine HCl increased the bulk lateral and rotational mobility of neuronal and model membrane lipid bilayes, and had a greater fluidizing effect on the inner monolayer than the outer monolayer. Lidocaine HCl increased annular lipid fluidity in SPMV lipid bilayers. It also caused membrane proteins to cluster. The most important finding of this study is that there is far greater increase in annular lipid fluidity than that in lateral and rotational mobilities by lidocaine HCl. Lidocaine HCl alters the stereo or dynamics of the proteins in the lipid bilayers by combining with lipids, especially with the annular lipids. In conclusion, the present data suggest that lidocaine, in addition to its direct interaction with proteins, concurrently interacts with membrane lipids, fluidizing the membrane, and thus inducing conformational changes of proteins known to be intimately associated with membrane lipid.

• Molecules and Cells
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• v.37 no.8
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• pp.592-597
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• 2014

The cell membrane provides critical cellular functions that rely on its elaborate structure and organization. The structure of turtle membranes is an important part of an ongoing study of erythrocyte membranes. Using a combination of atomic force microscopy and single-molecule force spectroscopy, we characterized the turtle erythrocyte membrane structure with molecular resolution in a quasi-native state. High-resolution images both leaflets of turtle erythrocyte membranes revealed a smooth outer membrane leaflet and a protein covered inner membrane leaflet. This asymmetry was verified by single-molecule force spectroscopy, which detects numerous exposed amino groups of membrane proteins in the inner membrane leaflet but much fewer in the outer leaflet. The asymmetric membrane structure of turtle erythrocytes is consistent with the semi-mosaic model of human, chicken and fish erythrocyte membrane structure, making the semi-mosaic model more widely applicable. From the perspective of biological evolution, this result may support the universality of the semi-mosaic model.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 1995.06b
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• pp.97-117
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• 1995

Copper resistance in Pseudomonas syringae pv. tomato is determined by copper-resistance operon (cop) on a highly conserved 35 kilobase plasmid. Copper-resistant strains of Pseudomonas syringae containing the cop operon accumulate copper and develop blue clonies on copper-containing media. The protein products of the copper-resistance operon were characterized to provide an understanding of the copper-resistance mechanism and its relationship to copper accumulation. The Cop proteins CopA (72 kDa), CopB (39 kDa), and CopC (12 kDa) were produced only under copper induction. CopA and CopC were periplasmic proteins and CopB was an outer membrane protein. Leader peptide sequences of CopA, CopB, and CopC were confirmed by amino-terminal peptide sequencing. CopA, CopB, and CopC were purified from strain PT23.2, and their copper contents were determined. One molecule of CopA bound 10.9${\pm}$1.2 atoms of copper and one molecule of CopC bound 0.6${\pm}$0.1 atom of copper. P. syringae cells containing copCD or copBCD cloned behind the lac promoter were hypersensitive to copper. The CopD (32 kDa), a probable inner membrane protein, function in copper uptake with CopC. The Cop proteins apparently mediate sequestration of copper outside of the cytoplasm as a copper-resistance mechanism.

• Journal of Ginseng Research
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• v.32 no.2
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• pp.99-106
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• 2008

A ligand - whether an endogenous hormone, neurotransmitter, exogenous toxin or synthetic drug - binds to plasma membrane proteins (e.g., ion channels, receptors or other functional proteins) to exert its physiological or pharmacological effects. Ligands can also have functional groups, showing stereospecificity for interaction sites on their counterpart plasma membrane proteins. Previous reports have shown that the ginsenoside Rg$_3$, a bioactive ginsenoside, meets these criteria in that: 1) an aliphatic side chain of $Rg_3$ plays a role as a functional group, 2) Rg$_3$ regulates voltage- and ligand-gated ion channels in a stereospecific manner with respect to carbon-20, and 3) $Rg_3$ regulates subsets of ligand-gated and voltage-gated ion channels through specific interactions with identified amino acid residues inside the channel pore, in the outer pore entryway, or in toxin binding sites. Rg$_3$, therefore, could be a candidate for a novel ginseng-derived glycosidic ligand regulating ion channels and receptors. This review will examine how Rg$_3$ regulates voltage-gated and ligand-gated ion channels through interactions with its target proteins in the plasma membrane. Hopefully, this review will advance understanding of ginseng pharmacology at the cellular and molecular levels.

• BMB Reports
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• v.37 no.5
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• pp.603-611
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• 2004

Fluorescence probes located in different membrane regions were used to evaluate the effects of chlorpromazine HCl on structural parameters (transbilayer lateral mobility, annular lipid fluidity, protein distribution, and lipid bilayer thickness) of synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortex. The experimental procedure was based on the selective quenching of 1,3-di(1-pyrenyl)propane (Py-3-Py) by trinitrophenyl groups, radiationless energy transfer from the tryptophan of membrane proteins to Py-3-Py, and energy transfer from Py-3-Py monomers to 1-anilinonaphthalene-8-sulfonic acid (ANS). In this study, chlorpromazine HCl decreased the lateral mobility of Py-3-Py in a concentration dependent-manner, showed a greater ordering effect on the inner monolayer than on the outer monolayer, decreased annular lipid fluidity in a dose dependent-manner, and contracted the membrane lipid bilayer. Furthermore, the drug was found to have a clustering effect on membrane proteins.

• Korean Chemical Engineering Research
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• v.60 no.3
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• pp.398-406
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• 2022

The outer membrane of Gram-negative bacteria is the outermost layer of cellular environment in which numerous biophysical and biochemical processes are in action sustaining viability. Advances in cell engineering enable modification of bacterial genetic information that subsequently alters membrane physiology to adapt bacteria to specific purposes. Surface display of a functional molecule on the outer membranes is one of strategies that directs host cells to respond to a specific extracellular matter or stimulus. While intracellular expression of a functional peptide or protein fused to a membrane-anchoring motif is commonly practiced for surface display, the method is not readily applicable to exogenous or large proteins inexpressible in bacteria. Chemical conjugation at reactive groups naturally occurring on the membrane might be an alternative, but often compromises fitness due to non-specific modification of essential components. Herein, we demonstrated two distinct approaches that enable site-specific decoration of the outer membrane with a fluorescent agent in Escherichia coli. An unnatural amino acid genetically incorporated in a surface-exposed peptide could act as a chemoselective handle for bioorthogonal dye labeling. A surface-displayed α-helical domain originating from a part of a selected heterodimeric coiled-coil complex could recruit and anchor a green fluorescent protein tagged with a complementary α-helical domain to the membrane surface in a site- and hetero-specific manner. These methods hold a promise as on-demand tools to confer new functionalities on the bacterial membranes.