• The Korean Journal of Nuclear Medicine
• /
• v.38 no.2
• /
• pp.190-197
• /
• 2004

Apoptosis plays a role in the pathophysiology of many kinds of diseases and in the response of treatment. Compared to the necrosis, the apoptosis is a genetically controlled and energy-dependent process which removes the unwanted cells from the body; programmed cell death or cell suicide. During the apoptosis, phosphatidylserine is expressed in the cytoplasmic outer membrane in the early phase. Annexin V, an endogenous human protein (MW=35 kD), has an affinity of about $10^{-9}\;M$ for the phosphatidylserine exposed on the outer membrane of apoptotic cells. Annexin V can be radiolabeled with $^{99m}Tc$ by HYNIC or EC chelators, which can be used as an radiotracer for the in vivo imaging of apoptosis. In this article, we reviewed the apoptosis, radiolabeling of annexin V, and the experimental and clinical data using annexin V imaging.

• Journal of Life Science
• /
• v.9 no.1
• /
• pp.35-39
• /
• 1999

Elecrophoreticl analysis of a 36 kDa protein was runned by SDS-PAGE, isoelectric focusing (IEF) and two dimensional electrophoresis pattern. Major 36 kDa and 25, 46, 48, 66 kDa protein were detected by Coomassie blue stain on SDS-PAGE. Major 36kDa protein was eluted for production of antiserum for serological analysis, IEF and two dimensional electrophoresis. Isoelectric point of 36kDa was aout pH 8.5. Two dimensional electrophoresis of eluted 36kDa showed one point on the gel. Anti-36 kDa serum made by newzilland rabbit for serological test. In ELISA, final titer of antibody was 100×{TEX}$2^5}${/TEX} : 1. Neutralize ability of serum was examined by slide agglutination test and colonization test in rat. Anti-36 kDa serum agglutinated whole cell of V. vulnificus were inhibited colonization on intestine in rat. Accordingly In this paper contain some electrophoretical analysis and serological test of a 36 kDa OMP of V. vulnificus.

• Journal of Periodontal and Implant Science
• /
• v.26 no.1
• /
• pp.133-142
• /
• 1996

Ten intrabony defects in 10 patients were treated by flap surgery including root surface debridement and placement of an expanded polytetrafluoroethylene(ePTFE) membrane. The membranes were removed after 4-6 weeks. This study was performed to examine the retrived ePTFE membrane by scanning electron microscopy(SEM) for bacterial contamination and adherent connective tissue elements, and to compare it with clinical conditions. The cervical portion of the membrane, which in most cases had become partially exposed to the oral cavity, had a bacterial deposit. Small bacterial colonies and a scatter of single cells in some instances extended into the apical portion of the membrane. Fibroblast-like cells, erythrocytes and fibrous structures were seen in the apical portion of the membrane. Outer surface of membrane tends to more bacterial contamination than inner surface(p<0.01), and upper portions more than lower portions(P<0.01). Comparison of ultrastructural findings and clinical conditions revealed that extent of bacterial contamination of the membrane correlated with gingival inflammation and extent of membrane exposure, but it was not significant statistically. The results suggested that gingival inflammation and membrane exposure affect periodontal regeneration by the use of ePTFE membrane.

• Proceedings of the Korean Society for Applied Microbiology Conference
• /
• 2001.06b
• /
• pp.14-16
• /
• 2001

The outer membrane (OM) of gram-negative bacteria acts as an effective permeability barrier against noxious agents including several antibiotics and organic solvents, and lipopolysaccharide (LPS) is the key molecule for this function. Outer membrane modified mutants (Ml-166, M2-42, M3-21) of E. coli DH5$\alpha$/pBSl were selected through a mutation using EMS (ethyl-methane-sulfonate). Among the selected mutants, M3-21 was twice as sensitive as LumisTo $x^{ }$ to benzene and M2-41 was 8 times as sensitive as LumisTo $x^{ }$ to toluene. To identify the structural change in the membrane by mutation, the relative cell surface hydrophobicities and the absorption of the crystal violet to the organisms were measured. All the mutants absorbed more crystal violet than their parent and the absorption of crystal violet increased in cell walls as carbohydrate of lipopolysaccharide decreased. When the cell surface hydrophobicities of DH5/pBSl and its mutants were measured by the BATH, the hydrophobicities of mutants increased compared to their parent in several organic solvents. The difference of lipopolysaccharide between DH5/pBSl and its mutants was identified by various ways such as the SDS-PAGE gel, the screening of LPS molecular weights, the mass spectrometry, and MALDI-TOF.F.

• Journal of Photoscience
• /
• v.8 no.3_4
• /
• pp.87-92
• /
• 2001

Intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py-3-Py) and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) were used to investigate the effects of parathyroid hormone (PTH) on the bulk bilayer fluidity of the plasma membrane vesicles isolated from cultured osteoblasts (OB-PMV). In a dose-dependent manner, rat PTH-(1-34) [rPTH-(1-34)] increased the excimer to monomer fluorescence intensity ratio (I'/I) of Py-3-Py and decreased the anisotropy (r) of DPH in OB-PMV. This indicates that PTH increased both the lateral and rotational diffusion of the probes in OB-PMY. Selective quenching of DPH fluorescence by trinitrophenyl groups was utilized to examine the transbilayer fluidity asymmetry of OB-PMV. The anisotropy, limiting anisotropy, and order parameter of DPH in the inner monolayer were 0.024, 0.032, and 0.062 greater than calculated for the outer monolayer of OB-PMY. Selective quenching of DPH fluorescence by trinitrophenyl groups was also utilized to examine the transbilayer effects of PTH on the fluidity of OB-PMV. rPTH-(1-34) had a greater fluidizing effect on the outer monolayer as compared to the inner monolayer of OB-PMV. Thus, it has been proven that PTH exhibits a selective rather than nonselective fluidizing effect within transbilayer domains of OB-PMV.

• Journal of Microbiology and Biotechnology
• /
• v.17 no.4
• /
• pp.594-599
• /
• 2007

It is well known that the Escherichia coli inner membrane-bound protease DegS is a periplasmic stress sensor for unfolded outer membrane proteins (OMPs). Previous studies have also shown that the outer membrane protease OmpT activates plasminogen in vitro and this may be exploited by bacteria in the course of pathogenesis. However, there has been no research on the plasminogen activation ability of the important periplasmic protein DegS. Accordingly, in this study, the whole-length and truncated degS genes were separately overexpressed in Escherichia coli, the recombinant proteins purified by affinity chromatography, and their plasminogen activator role tested in vitro. The results suggested that the whole-length DegS was able to activate plasminogen on a plasma plate. The truncated form of DegS (residues 80-345), designated ${\Delta}DegS$, also acted as a plasminogen activator, as confirmed by different assays. The serine protease property of ${\Delta}DegS$ was verified based on the complete inhibition of its enzyme activity by PMSF (phenylmethanesulfonyl fluoride). Therefore, the present results indicate that DegS is a plasminogen activator in vitro.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.2
• /
• pp.234-238
• /
• 2012

Recombinant Escherichia coli displaying organophosphorus hydrolase (OPH) was used to overcome the diffusion barrier limitation of organophosphorus pesticides. A new anchor system derived from the N-terminal domain of ice-nucleation protein from Pseudomonas syringae InaV (InaV-N) was used to display OPH onto the surface. The designed sequence was cloned in the vector pET-28a(+) and then was expressed in E. coli. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by InaV-N on the outer membrane, and the ability of recombinant E. coli to utilize diazinon as the sole source of energy, without growth inhibition, indicated its significant activity. The location of OPH was detected by comparing the activity of the outer membrane fraction with the inner membrane and cytoplasm fractions. Studies revealed that recombinant E. coli can degrade 50% of 2 mM chlorpyrifos in 2 min. It can be concluded that InaV-N can be used efficiently to display foreign functional protein, and these results highlight the high potential of an engineered bacterium to be used in bioremediation of pesticide-contaminated sources in the environment.

• Proceedings of the Korean Society of Plant Biotechnology Conference
• /
• 2005.04a
• /
• pp.119-119
• /
• 2005

• Journal of Microbiology and Biotechnology
• /
• v.27 no.4
• /
• pp.791-807
• /
• 2017

The type II secretion system (T2SS), which transports selected periplasmic proteins across the outer membrane, has rarely been studied in nonpathogens or in organisms classified as Betaproteobacteria. Therefore, we studied Cupriavidus metallidurans (Cme), a facultative chemilithoautotroph. Gel analysis of extracellular proteins revealed no remarkable differences between the wild type and the T2SS mutants. However, enzyme assays revealed that native extracellular alkaline phosphatase is a T2SS substrate, because activity was 10-fold greater for the wild type than a T2SS mutant. In Cme engineered to produce three Ralstonia solanacearum (Rso) exoenzymes, at least 95% of their total activities were extracellular, but unexpectedly high percentages of these exoenzymes remained extracellular in T2SS mutants cultured in rich broth. These conditions appear to permit an alternative secretion process, because neither cell lysis nor periplasmic leakage was observed when Cme produced a Pectobacterium carotovorum exoenzyme, and wild-type Cme cultured in minimal medium secreted 98% of Rso polygalacturonase, but 92% of this exoenzyme remained intracellular in T2SS mutants. We concluded that Cme has a functional T2SS despite lacking any abundant native T2SS substrates. The efficient secretion of three foreign exoenzymes by Cme is remarkable, but so too is the indication of an alternative secretion process in rich culture conditions. When not transiting the T2SS, we suggest that Rso exoenzymes are probably selectively packaged into outer membrane vesicles. Phylogenetic analysis of T2SS proteins supports the existence of at least three T2SS subfamilies, and we propose that Cme, as a representative of the Betaproteobacteria, could become a new useful model system for studying T2SS substrate specificity.

• Journal of Fisheries and Marine Sciences Education
• /
• v.27 no.5
• /
• pp.1508-1521
• /
• 2015

Vibrio harveyi is a pathogenic marine bacterium causing systemic symptoms resulting in mass mortalities in fishes and shrimps in aquaculture. Outer membrane proteins(OMPs) are related to the pathogenicity and thus good targets for diagnosis and vaccination for Gram negative bacteria. Recently vaccination strategies using the OMPs have been suggested to control vibriosis in several fish species. In this study, we have isolated V. harveyi from diseased marine fishes from different regions of Korea and investigated genetic variations of four OMP genes including OmpK, OmpU, OmpV and OmpW. Consequently, OmpK and U genes could be divided into 3 subgroups of type I, II, III and type A, B, C, respectively, without any correlation with geographical regions and species while OmpV and W were highly homologous. OmpW gene of V. harveyi FP4138 was fully sequenced and predicted the deduced amino acid sequence to form ${\beta}-barrel$ with hydrophobic channel. Indeed, the immunogenicity of recombinant OmpW produced in Escherichia coli was assessed by vaccinating flounder. As a result, the high antibody response with antibody titer of $4.2{\pm}0.7$ and protection with relative percent survival of 60% against artificial infection of V. harveyi were demonstrated. This result indicates that OmpW is a virulence related factor and it can be a vaccine candidate to prevent a high mortality caused by V. harveyi infection in olive flounder, Paralichthys olivaceus.