• Journal of Periodontal and Implant Science
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• v.43 no.1
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• pp.24-29
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• 2013

Purpose: Matrix metalloproteinases (MMPs) are capable of degrading extracellular matrix, and they are inducible enzymes depending on an inflammatory environment such as periodontitis and bacterial infection in periodontal tissue. Gingival inflammation has been postulated to be correlated with the production of MMP-2 and MMP-9. The objective of this study was to quantify the expression and activity of MMP-9 and -2, and to determine the correlation between activity and expression of these MMPs in human gingival tissues with periodontitis. Methods: The gingival tissues of 13 patients were homogenized in $500{\mu}L$ of phosphate buffered saline with a protease inhibitor cocktail. The expression and activity of MMP-2 and -9 were measured by enzyme-linked immunosorbent assay and Western blot analysis, and quantified by a densitometer. For the correlation line, statistical analysis was performed using the Systat software package. Results: MMP-9 was highly expressed in all gingival tissue samples, whereas MMP-2 was underexpressed compared with MMP-9. MMP-9 activity increased together with the MMP-9 expression level, with a positive correlation (r=0.793, P=0.01). The correlation was not observed in MMP-2. Conclusions: The expression of MMP-2 and -9 might contribute to periodontal physiological and pathological processes, and the degree of MMP-9 expression and activity are predictive indicators relevant to the progression of periodontitis.

• Journal of Microbiology and Biotechnology
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• v.9 no.2
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• pp.196-200
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• 1999

A recombinant human $\alpha_{s1}$-casein was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Three different leader sequences derived from the mating factor $\alpha$l (MF$\alpha$l), inulinase, and human $\alpha_{s1}$-casein were used to direct the secretion of human $\alpha_{s1}$-casein into the extracellular medium. Among the three leader sequences tested, the native leader sequence of human $\alpha_{s1}$-casein was found to be the most efficient in the secretory expression of human $\alpha_{s1}$-casein, which implies that the native leader sequence of human $\alpha_{s1}$-casein might be used very efficiently for the secretory production of other heterologous proteins in yeast. The recombinant human $\alpha_{s1}$-casein was proteolytically cleaved as the culture proceeded. Therefore, an attempt was made to produce human $\alpha_{s1}$-casein using a S. cerevisiae mutant in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted. After 72 h of culture, most of the human $\alpha_{s1}$-casein secreted by the wild type was cleaved, whereas more than 70% of the human $\alpha_{s1}$-casein secreted by yap3-disruptant remained intact. The results suggest that YAP3 might be involved in the internal cleavage of human $\alpha_{s1}$-casein expressed in yeast

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.3
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• pp.334-342
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• 2013

Proteases and protease inhibitors play key roles in most physiological processes, including cell migration, cell signaling, and cell surface and tissue remodeling. Among these, the matrix metalloproteinase (MMPs) pathway is one of the most efficient biosynthetic pathways for controlling the activation of enzymes responsible for protein degradation. This also indicates the association of MMPs with the maturation of spermatozoa. In an attempt to investigate the effect of MMP activation and inhibitors in cultures with various hormones during sperm capacitation, we examined and monitored the localization and expression of MMPs (MMP-2 and MMP-9), tissue inhibitors of metalloproteinases (TIMP-2 and TIMP-3), as well as their expression profiles. Matured spermatozoa were collected from cultures with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and Lutalyse at 1 h, 6 h, 18 h, and 24 h. ELISA detected the expression of MMP-2, MMP-9, TIMP-2, and TIMP-3 in all culture media, regardless of medium type (FSH-supplemented fertilization Brackett-Oliphant medium (FFBO), LH-supplemented FBO (LFBO), or Lutalyse-supplemented FBO (LuFBO)). TIMP-2 and TIMP-3 expression patterns decreased in LFBO and LuFBO. MMP-2 and MMP-9 activity in FBO and FFBO progressively increased from 1 h to 24 h but was not detected in LFBO and LuFBO. The localization and expression of TIMP-2 and TIMP-3 in sperm heads was also measured by immunofluorescence analysis. However, MMPs were not detected in the sperm heads. MMP and TIMP expression patterns differed according to the effect of various hormones. These findings suggest that MMPs have a role in sperm viability during capacitation. In conjunction with hormones, MMPs play a role in maintaining capacitation and fertilization by controlling extracellular matrix inhibitors of sperm.

• Journal of Life Science
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• v.21 no.12
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• pp.1716-1720
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• 2011

A bacterium hydrolysing various organic materials including cellulose, protein, starch and lipid was isolated. The isolate was identified as Bacillus subtilis, and named Bacillus subtilis CK-2 in this paper. This bacterium showed optimal growth at $40\sim45^{\circ}C$, pH 6~9, and 0~3% of NaCl. B. subtilis CK-2 seemed to synthesis highly active autolysin. The hydrolytic enzymes produced by B. subtilis CK-2 were primary enzymes because extracellular enzyme activities varied similarly to the growth curve. The hydrolytic enzymes seemed to be stable at basic pH conditions. From these results, B. subtilis CK-2 was found to bea useful bacterial agent for composting, or for use in feed-production waste in agriculture, fishery, forest materials, livestock farming, and food.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.12
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• pp.1844-1853
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• 2019

Objective: We investigated how pituitary adenylate cyclase-activating polypeptide (PACAP) affects embryonic development during pre-in vitro maturation (pre-IVM) using porcine oocytes isolated from small follicles. Methods: We divided the follicles into the experimental groups by size (SF, small follicles; MF, medium follicles) and treated with and without PACAP and cultured for 18 hours (PreSF[-]PACAP; without PACAP, Pre-SF[+]PACAP; with PACAP) before undergoing IVM. The gene expression related to extracellular matrix formation (amphiregulin, epiregulin, and hyaluronan synthase 2 [HAS2]) and apoptosis (Bcl-2-associated X [BAX], B-cell lymphoma 2, and cysteine-aspartic acid protease 3) was investigated after maturation. The impact on developmental competence was assessed by the cleavage and blastocyst rate and total cell number of blastocysts in embryos generated from parthenogenesis (PA) and in vitro fertilization (IVF). Results: Cleavage rates in the Pre-SF(+)PACAP after PA were significantly higher than SF and Pre-SF(-)PACAP (p<0.05). The cleavage rates between MF and Pre- SF(+)PACAP groups yielded no notable differences after IVF. Pre-SF(+)PACAP displayed the higher rate of blastocyst formation and greater total cell number than SF and Pre-SF(-)PACAP (p<0.05). Cumulus cells showed significant upregulation of HAS2 mRNA in the Pre-SF(+)PACAP compared to the SF (p<0.05). In comparison to other groups, the Pre-SF(+)PACAP group displayed a downregulation in mRNA expression of BAX in matured oocytes (p<0.05). Conclusion: The PACAP treatment during pre-IVM improved the developmental potential of porcine oocytes derived from SF by regulating cumulus expansion and apoptosis of oocytes.

• KSBB Journal
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• v.31 no.4
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• pp.277-283
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• 2016

In this system, rice cells were genetically modified to express human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) using RAmy3D promoter induced by sugar depletion. Even though the target protein fused with signal sequence peptide, plant cell wall can be a barrier against secretion of recombinant proteins. Therefore, hCTLA4Ig can be trapped inside cell wall or remained in intracellular space. In this study, to enhance the secretion of hCTLA4Ig from cytoplasm and cell walls into the medium, permeabilizing agents, such as dimethyl sulfoxide (DMSO), Triton X-100 and Tween 20, were applied in transgenic rice cell cultures. When 0.5% (v/v) of DMSO was added in sugar-free medium, intracellullar hCTLA4Ig was increased, on the other hand, the secreted extracellular hCTLA4Ig was lower than that of control. DMSO did not give permeable effects on transgenic rice cell cultures. And Triton X-100 was toxic to rice cells and also did not give enhancing permeability of cells. When 0.05% (v/v) Tween 20 was added in rice cell cultures, however, intracellular hCTLA4Ig was lower than that of control cultures. And the maximum 44.76 mg/L hCTLA4Ig was produced for 10 days after induction, which was 1.4-fold increase compared to that of control cultures. Especially, Tween 20 at 0.05% (v/v) showed the positive effect on the secretion of hCTLA4Ig though the decrease of intracellular hCTLA4Ig. Also, Tween 20 as a non-toxic surfactant did not affect the cell growth, cell viability and protease activity. In conclusion, secretion of hCTLA4Ig could be increased by enhancing permeability of cells regardless of the cell growth, cell viability and protease activity.

• Journal of Life Science
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• v.22 no.6
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• pp.823-827
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• 2012

A bacterial strain producing a fibrinolytic enzyme, subtilisin CP-1, was isolated from Doen-Jang, a Korean traditional fermentation food. Based on the analysis of gene sequence of 16S rRNA and biochemical analysis, the strain was identified as Bacillus sp. and named as Bacillus sp. CP-1. To investigate the effect of the medium on the production of fibrinolytic enzyme from Bacillus sp. CP-1, two commercial bacterial culture media, tryptic soy broth (TSB) and Luria-Bertani (LB), were applied to the cultivation of Bacillus sp. CP-1. The strain secreted only one proteolytic enzyme (subtilisin CP-1) in the culture broth. The molecular weight of subtilisin CP-1 was estimated to be 28 kDa. Subtilisin CP-1 was optimally active at pH 9.0 and $45^{\circ}C$, and exhibited high specificity for Meo-Suc-Arg-Pro-Tyr-pNA (S-2586), a synthetic chromogenic substrate for chymotrypsin. The first eight amino acid residues of the N-terminal sequence of the enzyme are AQSVPYGI; this sequence is identical to that of subtilisin NAT and E.

• Journal of Life Science
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• v.15 no.4 s.71
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• pp.657-663
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• 2005

Collagenases are generally defined as enzymes that are capable of degrading the polypeptide backbone of native collagen under conditions that do not denature the protein. An extracellular collagenase-producing bacterial strain was isolated from kimchi and identified to be Bacillus subtilis JS-17 through morphological, cultural, biochemical characteristics and 16S rDNA sequence analysis. Optimum culture condition of Bacillus subtilis JS-17 for the production of collagenase was $1.5\%$ fructose, $1\%$ yeast extract, $0.5\%\;K_2HPO_4,\;0.4\%\;KH_2PO_4,\;0.01\%\;MgSO_4\cdot7H_2O,\;0.01\%\; MnSO_4\cdot4H_2O,\;,0.1\%$ citrate and $0.1\%\;CaCl_2$. The production of collagenase was optimal at $30^{\circ}C$ for 72 hr. A collagenase was isolated from the culture filtrate of Bacillus subtilis JS-17. The enzyme was purified using Amberlite IRA-900 column chromatography, Sephacryl S-300 HR column chromatography and DEAE-Sephadex A-50 column chromatography The purified collagenase has an specific activity 192.1 units/mg. The molecular weight of the purified enzyme was estimated to be 28 kDa by SDS-PACE. The purified collagenase has $100\%$ activity up to $55^{\circ}C$.

• Korean Journal of Soil Science and Fertilizer
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• v.45 no.6
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• pp.1126-1135
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• 2012

In order to produce high-quality fermenting composts, bacteria strains with high activities of extracellular enzymes (cellulase, chitinase, amylase, protease and lipase) were isolated from the soils in 6 provinces of Korea, and characterized by 16S rRNA gene sequence analysis and properties. The selected 7 stains inoculated to livestock manure for 2' fermenting time, and experimental treatment divided into 3 groups, B1, B2 and B3, according to microbial activity and enzyme type. Our results showed that microbe applications (B1, B2 and B3) can increase (p<0.05) both rhizomes (17-38%) and enzyme activities (50-81%) in compost after fermenting time, respectively, compared to non-microbe treatment (control). The microbe application also decreased significantly (p<0.05) the $NH_3$ and $H_2S$ gas contents 13.4 and 27.3% compared with control, and the Propionic acid and Butyric acid gas contents 14.5 and 19.6%, respectively, as compared to the control. The microbial degradation rate (%) of pesticides and heavy metals increased significantly (p<0.05) after fermenting time, respectively, as compared to the control. Especially, microbe applications were more effective in total rhizomes yields and bioactivities than non-microbe treatment. Thus the results of this study could help in development of potential bioinoculants and composting techniques that maybe suitable for crop production, and protectable for earth environment under various conditions.