• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.305-311
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• 2002

Acetyl xylan esterase gene (AXE) from Aspergillus ficuum was cloned and its Pichia expression plasmid, pPICZ$\alpha$C-AXE (4.6 kb), was constructed, in which the AXE gene was under the control of the AOXI promoter and connected downstream of mating factor u-1 signal sequence. The plasmid linearized by Sacl was integrated into the 5'AOXI region of the chromosomal DNA of P. pastoris. In the flask batch culture of P. pastoris transformant on methanol medium, the cell concentration and total AXEase activity reached at 6.0 g-dry cell weight/1 and 77 unit/ml after 36 h cultivation, respectively. In the fed-batch culture employing the optimized methanol and histidine feeding strategy, the cell concentration and total AXEase activity were significantly increased to about 97 g-dry cell weight/1 and 930 unit/ml. Most of AXEase activity (>90%) was found in the extracellular medium and the majority of extracellular protein (>80%) was AXEase enzyme (33.5 kDa). This result means that about 9.8 g/1 of AXEase protein was produced in the extracellular medium.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.254-264
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• 2010

To obtain an efficient natural lignocellulolytic complex enzyme, we screened an efficient lignocellulose-degrading composite microbial system (XDC-2) from composted agricultural and animal wastes amended soil following a long-term directed acclimation. Not only could the XDC-2 degrade natural lignocelluloses, but it could also secrete extracellular xylanase efficiently in liquid culture under static conditions at room temperature. The XDC-2 degraded rice straw by 60.3% after fermentation for 15 days. Hemicelluloses were decomposed effectively, whereas the extracellular xylanase activity was dominant with an activity of 8.357 U/ml on day 6 of the fermentation period. The extracellular crude enzyme noticeably hydrolyzed natural lignocelluloses. The optimum temperature and pH for the xylanase activity were $40^{\circ}C$ and 6.0. However, the xylanase was activated in a wide pH range of 3.0-10.0, and retained more than 80% of its activity at $25-35^{\circ}C$ and pH 5.0-8.0 after three days of incubation in liquid culture under static conditions. PCR-DGGE analysis of successive subcultures indicated that the XDC-2 was structurally stable over long-term restricted and directed cultivation. Analysis of the 168 rRNA gene clone library showed that the XDC-2 was mainly composed of mesophilic bacteria related to the genera Clostridium, Bacteroides, Alcaligenes, Pseudomonas, etc. Our results offer a new approach to exploring efficient lignocellulolytic enzymes by constructing a high-performance composite microbial system with synergistic complex enzymes.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.6
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• pp.485-489
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• 2015

Diabetic nephropathy (DN) is the leading cause of end-stage failure of the kidney, but the efficacy of currently available strategies for the prevention of DN remains unsatisfactory. In this study, we investigated the effects of free anthraquinones (FARs) extract, which was extracted from the rhubarb and purified by macroporous resin DM130 with gradient mixtures of ethanol/water as the lelution solvents, in high glucose-cultured glomerular mesangial cells (MCs). The cell proliferation was determined by CCK-8 assay, the levels of TGF-${\beta}1$, CTGF, ColIV and FN proteins in the supernatant of MCs were measured by ELISA assays, and the mRNA levels of these four genes were detected by RT-PCR. The results showed that the increased proliferation of MCs, the mRNA levels and protein expression of TGF-${\beta}1$, CTGF, ColIV and FN induced by high glucose were inhibited after the treatment with the FARs extract. This indicated that FARs extract could inhibit cell proliferation and the expression of main extracellular matrix induced by high glucose in MCs. The FARs extract exhibited potential values for prophylaxis and therapy of DN.

• The Journal of Advanced Prosthodontics
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• v.6 no.5
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• pp.351-360
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• 2014

PURPOSE. The aim of present study was to identify characteristic and response of mouse bone marrow (BM) derived low-adherent bone marrow mesenchymal stem cells (BMMSCs) obtained by quantification of extracellular matrix (ECM). MATERIALS AND METHODS. Non-adherent cells acquired by ECM coated dishes were termed low-adherent BMMSCs and these cells were analyzed by in vitro and in vivo methods, including colony forming unit fibroblast (CFU-f), bromodeoxyuridine (BrdU), multi-potential differentiation, flow cytometry and transplantation into nude mouse to measure the bone formation ability of these low-adherent BMMSCs. Titanium (Ti) discs with machined and anodized surfaces were prepared. Adherent and low-adherent BMMSCs were cultured on the Ti discs for testing their proliferation. RESULTS. The amount of CFU-f cells was significantly higher when non-adherent cells were cultured on ECM coated dishes, which was made by 7 days culturing of adherent BMMSCs. Low-adherent BMMSCs had proliferation and differentiation potential as adherent BMMSCs in vitro. The mean amount bone formation of adherent and low-adherent BMMSCs was also investigated in vivo. There was higher cell proliferation appearance in adherent and low-adherent BMMSCs seeded on anodized Ti discs than machined Ti discs by time. CONCLUSION. Low-adherent BMMSCs acquired by ECM from non-adherent cell populations maintained potential characteristic similar to those of the adherent BMMSCs and therefore could be used effectively as adherent BMMSCs in clinic.

• Biomedical Science Letters
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• v.15 no.4
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• pp.319-326
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• 2009

Extracellular ATP elicits diverse physiological effects by binding to the G-protein-coupled P2Y receptors on the plasma membrane. In addition to the short-term effects of extracellular nucleotides on cell functions, there is evidence that such purinergic signalling can have long-term effects on cell proliferation, differentiation and death. The 3T3-L1 cell line derived from mouse embryo is a well-established and commonly utilized in vitro model for adipocytes differentiation and function. However, the distributions and roles of P2Y subtypes are still unknown in the preadipocyte. In this study, we identified the distributions and roles of P2Y subtypes in preadipocyte using $Ca^{2+}$ imaging and realtime PCR. ATP increased the $[Ca^{2+}]_i$ in a concentration-dependent manner. ATP increased $Ca^{2+}$ in absence and/or presence of extracellular $Ca^{2+}$. Suramin, non-selective P2Y blocker, largely blocked the ATP-induced $Ca^{2+}$ response. U73122, a PLC inhibitor, completely inhibited $Ca^{2+}$ mobilization in 3T3-L1 cells. The mRNA expression by realtime PCR of P2Y subtypes was $P2Y_2:P2Y_5:P2Y_6=1.0:12.5:0.3$. In conclusion, we showed that $P2Y_5$ receptor is a dominant purinergic receptor in preadipocytes, and multiple P2Y receptors could involve in differentiation and migration via regulating of intracellular calcium concentration.

• Molecules and Cells
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• v.41 no.3
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• pp.179-187
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• 2018

Proteomic analysis of extracellular vesicles (EVs) from biological fluid is a powerful approach to discover potential biomarkers for human diseases including cancers, as EV secreted to biological fluids are originated from the affected tissue. In order to investigate significant molecules related to the pathogenesis of bladder cancer, EVs were isolated from patient urine which was analyzed by mass spectrometry based proteomics. Comparison of the EV proteome to the whole urine proteome demonstrated an increased number of protein identification in EV. Comparative MS analyses of urinary EV from control subjects and bladder cancer patients identified a total of 1,222 proteins. Statistical analyses provided 56 proteins significantly increased in bladder cancer urine, including proteins for which expression levels varied by cancer stage (P-value < 0.05). While urine represents a valuable, non-invasive specimen for biomarker discovery in urologic cancers, there is a high degree of intra- and inter-individual variability in urine samples. The enrichment of urinary EV demonstrated its capability and applicability of providing a focused identification of biologically relevant proteins in urological diseases.

• Journal of Periodontal and Implant Science
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• v.47 no.6
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• pp.363-371
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• 2017

Purpose: The purpose of this study was to investigate the feasibility of regenerative therapy with a collagenated bone graft and resorbable membrane in intrabony defects, and to evaluate the effects of the novel extracellular matrix (ECM)-based membrane clinically and radiologically. Methods: Periodontal tissue regeneration procedure was performed using an ECM-based resorbable membrane in combination with a collagenated bovine bone graft in intrabony defects around the teeth and implants. A novel extracellular matrix membrane (NEM) and a widely-used membrane (WEM) were randomly applied to the test group and the control group, respectively. Cone-beam computed tomography images were obtained on the day of surgery and 6 months after the procedure. Alginate impressions were taken and plaster models were made 1 week and 6 months postoperatively. Results: The quantity of bone tissue, the dimensional changes of the surgically treated intrabony defects, and the changes in width and height below the grafted bone substitutes showed no significant difference between the test and control groups at the 6-month examination. Conclusions: The use of NEM for periodontal regeneration with a collagenated bovine bone graft showed similar clinical and radiologic results to those obtained using WEM.

• BMB Reports
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• v.34 no.6
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• pp.509-516
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• 2001

A genomic DNA fragment that contains the gene, which codes for a novel extracellular serine protease in Burkholderia pseudomallei, was cloned by using pQE40 as a vector. It was maintained in Escherichia coli JM109. The expression of the gene(s) resulted in the production of a 52 kDa protease. The recombinant protease was purified from the culture filtrate via ammonium sulfate fractionation, gel filtration, and anion-exchange chromatography. The purified protease had an optimum pH and temperature of pH 8.9 and $38^{\circ}C$, respectively. The protease activity was inhibited by EGTA, EDTA, and PMSF, but not 1,10-phenanthroline. The first 11 amino acid residues from the N-terminus of the purified protease were identified as LAPNDPYYYGY. PNDPYY was found to show homology to the Bacillus cereus microbial serine protease and B. subtilis PD498 serine protease. These results indicate that the protease that was purified in this study is an extracellular calcium-dependent serine protease. The purified protease was able to digest the human serum 19A, IgG, albumin, and transferrin, as well as bovine muscle actin and myosin. Furthermore, it was able to promote or cause dermonecrosis in experimental rabbits. These results propose the possible role of a novel B. pseudomallei extracellular calcium-dependent serine protease in the virulence of the pathogen.

• BMB Reports
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• v.29 no.5
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• pp.405-410
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• 1996

Previous studies have shown that transforming growth factor beta ($TGF-{\beta}$) is a potent regulator of cell growth and differentiation. To study the effects of $TGF-{\beta}$ on cell morphology and cytoskeleton reorganization, we conducted a survey using Mv1Lu mink lung epithelial cells with antibodies to cytoskeletal proteins and an extracellular matrix protein. While the untreated cells showed a cuboidal shape of typical epithelia, the Mv1Lu cells displayed a drastic shape change in the presence of $TGF-{\beta}$. This alteration was most prominent when near-confluent cells were treated with $TGF-{\beta}$. Since the morphology alteration is known to be accompanied by the reorganization of cytoskeletal proteins in other cell types, we investigated the intracellular distribution of the three major cytoskeletal structures: microfilaments, microtubules, and intermediate filaments. In the microfilament system, $TGF-{\beta}$ induced new stress fiber formation, which was caused primarily by the polymerization of cytoplasmic G-actin. However, $TGF-{\beta}$ appeared not to induce any significant changes in microtubular structures and vimentin filaments as determined by indirect fluorescence microscopy. Finally we confirmed the rapid accumulation of fibronectin by immunoblot analysis and chased the protein locations by immunofluorescence microscopy.

• Journal of the Korea Organic Resources Recycling Association
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• v.27 no.4
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• pp.43-49
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• 2019

In order to investigate the relation between sewage sludge solubilization and extracellular polymeric substances(EPS) during alkaline-ultrasonic pretreatment, SCOD/TCOD ratio, solubilization rate, VSS/TS ratio, VSS reduction rate, LB-EPS(Loosely-Bound EPS) and TB-EPS(Tightly-Bound EPS) were measured. At the condition of TS 1.0% and pH 12, solubilization rate increased by 27.7%, LB-EPS as Carbohydrate and Protein increased by 14.6 and 13.3 mg/L/g TS, respectively. Withal, VSS decreased by 26.7% and TB-EPS as Carbohydrate and Protein were extracted by 15.7 and 21.9 mg/L/g TS, respectively. Consequently, the concentrations of organic matter and LB-EPS increased and the trends appeared similarly. In addition, the concentrations trend of decreasing solid matter and extracted TB-EPS also appeared similarly.