• Journal of Korean Dental Science
• /
• v.11 no.2
• /
• pp.62-70
• /
• 2018

Purpose: Bone marrow has long been a source of primary cells. This study was performed to evaluate the effects of age and sex on the cellular viability and expression of stem cell markers of mRNA and on the protein expression of bone marrow stem cells (BMSCs) derived from healthy donors. Materials and Methods: Stem cells were isolated from human bone marrow and plated in culture plates. The shape of the BMSCs was observed under inverted microscope. Quantitative cellular viability was evaluated using a Cell-Counting Kit-8 assay. The expression of stem cell surface markers was tested and a series of quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot was performed to evaluate the expression in each group. Result: The shapes of the cells at 20s, 30s, and 50s were similar to each other. No significant changes in cellular viability were noted among different age groups or sex groups. The BMSCs expressed CD44, CD73, and CD90 surface markers but did not express CD14 and CD34. There were no noticeable differences in CD surface markers among the different age groups. The expressions of CD surface markers were similar between men and women. No significant differences in the secretion of vascular endothelial growth factors (VEGFs) were noted at Day 3 between different age groups. qRT-PCR regarding the expression showed differences between the age groups. However, Western blot analysis showed a decrease in expression but did not reach statistical significance (P>0.05). Conclusion: This study clearly showed no significant differences in shape, cell viability, expression of stem cell surface markers, or secretion of human VEGF among different age groups. However, western blot analysis showed a tendency of age-related decrease which did not reach statistical significance. Collectively, autologous or allogeneic BMSCs should be meticulously applied to obtain optimal results regarding age and sex.

• Biomedical Science Letters
• /
• v.14 no.4
• /
• pp.203-210
• /
• 2008

One of cell surface receptor proteins, human folate receptor (hFR) involves in the uptake of folates through cell membrane into cytoplasm, and is anchored to the plasma membrane by a fatty acid linkage, which has been identified in some cells as a glycosylphosphatidylinositol (GPI)-tailed protein with a molecular mass of about 40 kDa. The hFR is released by phosphatidylinositol phospholipase C (PI-PLC) because it contains fatty acids and inositol on the GPI tail. Caveolin decorates the cytoplasmic surface of caveolae and has been proposed to have a structural role in maintaining caveolae. It is unknown whether caveolin is involved in targeting, and is necessary for the function of GPI-tailed proteins. To compare the ability of folic acid binding, internalization and expression of hFR, and the effect of caveolin at the both apical and basolateral side of cell surfaces in Chinese hamster ovary (CHO) clone cells overexpressed the hFR and/or caveolin. Our present results suggest a possibility that the overexpression of caveolin does not be involved in expression of hFR, but plays a role as a factor in PI-PLC releasing kinetics, and for a regulation of formation, processing and function of hFR in CHO clone cells overexpressed cavcolin.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.12
• /
• pp.1330-1335
• /
• 2011

N-Acyl homoserine lactones (AHLs) serve as the vital quorum-sensing signals that regulate the virulence of the pathogenic bacterium Erwinia carotovora. In the present study, an approach to efficiently restrain the pathogenicity of E. carotovora-induced soft rot disease is described. Bacillus thuringiensis-derived N-acyl homoserine lactonase (AiiA) was projected onto the surface of Pseudomonas putida cells, and inoculation with both strains was challenged. The previously identified N-terminal moiety of the ice nucleation protein, InaQ-N, was applied as the anchoring motif. A surface display cassette with inaQ-N/aiiA was constructed and expressed under the control of a constitutive promoter in P. putida AB92019. Surface localization of the fusion protein was confirmed by Western blot analysis, flow cytometry, and immunofluorescence microscopy. The antagonistic activity of P. putida MB116 expressing InaQ-N/AiiA toward E. carotovora ATCC25270 was evaluated by challenge inoculation in potato slices at different ratios. The results revealed a remarkable suppressing effect on E. carotovora infection. The active component was further analyzed using different cell fractions, and the cell surface-projected fusion protein was found to correspond to the suppressing effect.

• Journal of Microbiology and Biotechnology
• /
• v.30 no.7
• /
• pp.1097-1103
• /
• 2020

Bacterial surface display systems have been developed for various applications in biotechnology and industry. Particularly, the discovery and design of anchoring motifs is highly important for the successful display of a target protein or peptide on the surface of bacteria. In this study, an efficient display system on Escherichia coli was developed using novel anchoring motifs designed from the E. coli mipA gene. Using the C-terminal fusion system of an industrial enzyme, Pseudomonas fluorescens lipase, six possible fusion sites, V¹⁴⁰, V¹⁷⁶, K¹⁷⁹, V²²⁶, V²³², and K²³⁴, which were truncated from the C-terminal end of the mipA gene (MV¹⁴⁰, MV¹⁷⁶, MV¹⁷⁹, MV²²⁶, MV²³², and MV²³⁴) were examined. The whole-cell lipase activities showed that MV¹⁴⁰ was the best among the six anchoring motifs. Furthermore, the lipase activity obtained using MV¹⁴⁰ as the anchoring motif was approximately 20-fold higher than that of the previous anchoring motifs FadL and OprF but slightly higher than that of YiaTR232. Western blotting and confocal microscopy further confirmed the localization of the fusion lipase displayed on the E. coli surface using the truncated MV¹⁴⁰. Additionally the MV¹⁴⁰ motif could be used for successfully displaying another industrial enzyme, α-amylase from Bacillus subtilis. These results showed that the fusion proteins using the MV¹⁴⁰ motif had notably high enzyme activities and did not exert any adverse effects on either cell growth or outer membrane integrity. Thus, this study shows that MipA can be used as a novel anchoring motif for more efficient bacterial surface display in the biotechnological and industrial fields.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.11
• /
• pp.1835-1841
• /
• 2015

A cell surface display system for heterologous expression of the multifunctional cellulase, CelEx-BR12, in Escherichia coli was developed using truncated E. coli outer membrane protein C (OmpC) as an anchor motif. Cell surface expression of CelEx-BR12 cellulase in E. coli harboring OmpC-fused CelEx-BR12, designated MC4100 (pTOCBR12), was confirmed by fluorescence-activated cell sorting and analysis of outer membrane fractions by western blotting, which verified the expected molecular mass of OmpC-fused CelEx-BR12 (~72 kDa). Functional evidence for exocellulase activity was provided by enzymatic assays of whole cells and outer membrane protein fractions from E. coli MC4100 (pTOCBR12). The stability of E. coli MC4100 (pTOCBR12) cellulase activity was tested by carrying out repeated reaction cycles, which demonstrated the reusability of recombinant cells. Finally, we showed that recombinant E. coli cells displaying the CelEx-BR12 enzyme on the cell surface were capable of growth using carboxymethyl cellulose as the sole carbon source.

• Polymer(Korea)
• /
• v.28 no.6
• /
• pp.502-508
• /
• 2004

Cationic liposomes for cancer treatment have been developed in the field of chemotharpy. It was well combined on the surface of anionic tumor cell membrane by electrostatic interaction. Thus, the object of this study was to prepare the cationic liposomes capable of forming an ionic complex with the anionic cell membrane. To prepare the cationic liposomes, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) as a cationic lipid material and polyethylenimine (PEI) as a cationic polymer were synthesized. Ionic property on the surface of liposomes was determined by the zeta potential. The adsorption characteristics of plasma protein for liposome in bovine serum were determined by the particle size and turbidity change. To estimate the stability of liposome in buffered solution, the change of particle size was measured at room temperature for seven days. The cationic liposomes were absorbed a large amount of plasma protein in bovine serum because plasma protein having anionic charge was fixed on the surface of cationic liposomes. This result indicate that the modification on the surface of liposomes using cationic polyethylenimine enhances the protein adsorption in bovine serum. Additionaly, cationic liposomes showed good stability in buffered solution for seven days.

• KSBB Journal
• /
• v.26 no.3
• /
• pp.199-205
• /
• 2011

Using tetrameric ${\beta}$-galactosidase as a model protein, anchoring motives were screened in Bacillus subtilis spore display system. Eleven spore coat proteins were selected considering their expression levels and the location in the spore coat layer. After chromosomal single-copy homologous integration in the amyE site of Bacillus subtilis chromosome, cotE and cotG were chosen as possible spore surface anchoring motives with their higher whole cell ${\beta}$-galactosidase activity. PAGE and Wester blot of extracted fraction of outer layer of purified spore, which express CotE-LacZ or CotG-LacZ fusion verified the existence of exact size of fusion protein and its location in outer coat layer of purified spore. ${\beta}$-galactosidase activity of spore with CotE-LacZ or CotG-LacZ fusion reached its highest value around 16~20 h of culture time in terms of whole cell and purified spore. After intensive spore purification with lysozyme treatment and renografin treatment, spore of BJH135, which expresses CotE-LacZ, retained only 1~2% of its whole cell ${\beta}$-galactosidase activity. Whereas spore of BJH136, which has cotG-lacZ cassette in the chromosome, retained 10~15% of its whole cell ${\beta}$-galactosidase activity, proving minor perturbation of CotG-LacZ, when incorporated in the spore coat layer of Bacillus subtilis compared to CotE-LacZ. Usage of Bacillus subtilis WB700, of which 7 proteases are knocked-out and thereby resulting in 99.7% decrease in protease activity of the host, did not prevent the proteolytic degradation of spore surface expressed CotG-LacZ fusion protein.

• Mycobiology
• /
• v.38 no.2
• /
• pp.108-112
• /
• 2010

To investigate the possible roles of LAMMER kinase homologue, Lkh1, in Schizosaccharomyces pombe, whole proteins were extracted from wild type and lkh1-deletion mutant cells and subjected to polyacrylamide gel electrophoresis. Differentially expressed proteins were identified by tandem mass spectrometry (MS/MS) and were compared with a protein database. In whole-cell extracts, 10 proteins were up-regulated and 9 proteins were down-regulated in the mutant. In extracellular preparations, 6 proteins were up-regulated in the lkh1+ null mutant and 4 proteins successfully identified: glycolipid anchored surface precursor, $\beta$-glucosidase (Psu1), cell surface protein, glucan 1,3-$\beta$-glucosidase (Bgl2), and exo-1,3 $\beta$-glucanase (Exg1). These results suggest that Lkh1 is involved in regulating cell wall assembly.

• IMMUNE NETWORK
• /
• v.1 no.1
• /
• pp.14-25
• /
• 2001

Background: Bone marrow stromal cells (BMSCs) express many cell surface molecules, which regulate the proliferation and differentiation of immune cells within the bone marrow. Methods: To identify cell surface molecules, which can regulate cell proliferation through cell interaction, monoclonal antibodies (MoAbs) against BMSCs were produced. Among them, 1H8 MoAb, which recognized distinctly an 80 kDa protein, abolished myeloma cell proliferation that was induced by co-culturing with BMSCs. Results: IL-6 gene expression was increased when myeloma or stromal cells were treated with 1H8 MoAb. In addition, the expression of IL-6 receptor and CD40 was up-regulated by 1H8 treatment, suggesting that the molecule recognized by 1H8 MoAb is involved in cell proliferation by modulating the expression of cell growth-related genes. Myeloma cells contain high levels of reactive oxygen species (ROS), which are related to gene expression and tumorigenesis. Treatment with 1H8 decreased the intracellular ROS level and increased PAG antioxidant gene concomitantly. Finally, 1H8 induced the tyrosine phosphorylation of several proteins in U266. Conclusion: Taken together, 1H8 MoAb recognized the cell surface molecule and triggered the intracellular signals, which led to modulate gene expression and cell proliferation.

• Bulletin of the Korean Chemical Society
• /
• v.29 no.5
• /
• pp.1013-1017
• /
• 2008

Syndecan-3 is a cell-surface heparan sulfate proteoglycan, which performs a variety of functions during cell adhension process. It is also a coreceptor for growth factor, mediating cell-cell and cell-matrix interaction. Syndecan-3 contains a cytoplasmic domain potentially associated with the cytoskeleton. Syndecan-3 is specifically expressed in neuron cell and has related to neuron cell differentiation and development of actin filament in cell migration. Syndecans each have a unique, central, and variable (V) region in their cytoplasmic domains. And that region of syndecan-3 may modulate the interactions of the conserved C1 regions of the cytoplasmic domains by tyrosine phosphorylation. Cytoplasmic domain of syndecan-3 has been synthesized for NMR structural studies. The solution structure of syndecan-3 cytoplasmic domain has been determined by two-dimensional NMR spectroscopy and simulated-annealing calculation. The cytoplasmic domain of the syndecan proteins has a tendency to form a dimmer conformation with a central cavity, however, that of syndecan-3 demonstrated a monomer conformation with a flexible region near C-terminus. The structural information might add knowledge about the structure-function relationships among syndecan proteins.