• Journal of Life Science
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• v.27 no.2
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• pp.202-210
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• 2017

As an effort to find a potential biopreservative, we isolated bacterial strains producing bacteriocin from fermented foods. A strain was finally selected and characteristics of the bacteriocin were investigated. The selected strain was identified as Bacillus subtilis E9-1 based on the 16S rRNA gene analysis. The culture supernatant of B. subtilis E9-1 showed antimicrobial activity against Gram-positive bacteria. Subtilisin A, ${\alpha}$-chymotrypsin, trypsin and proteinase K inactivated the antimicrobial activity, which means its proteinaceous nature, a bacteriocin. The bacteriocin activity was fully retained at the pH range from 2.0 to 8.0 and stable at up to $100^{\circ}C$ for 60 min. Solvents such as ethanol, isopropanol and methanol had no effect on the antimicrobial activity at the concentration of 100% but acetone and acetonitrile reduced the activity at up to 100% concentration. Cell growth of four indicator strains was dramatically decreased in dose-dependent manner. Listeria monocytogenes was the most sensitive, but Enterococcus faecium was the most resistant. Bacillus cereus and Staphylococcus aureus showed the medium sensitivity. The bacteriocin showed its antimicrobial activity against B. cereus and L. monocytogenes via bactericidal action. The number of viable cells of L. monocytogenes started to reduce after addition of bacteriocin to the minced beef. The bacteriocin was purified through acetone concentration, gel filtration chromatography and RP-HPLC. The whole purification step led to a 6.82 fold increase in the specific activity and 6% yield of bacteriocin activity. The molecular weight of the purified bacteriocin was determined to be 3.3 kDa by MALDI-TOF/TOF mass spectrometry.

• Journal of Gastric Cancer
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• v.4 no.4
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• pp.207-212
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• 2004

Purpose: Invasion and metastasis in solid tumors require the action of tumor-associated proteases. The serine protease urokinase-type plasminogen (uPA) and receptor (uPAR) appear to have a major function in these processes. Expression of the uPAR is elevated in breast and colon carcinomas, and this is often associated with invasiveness and poor prognosis. The purpose of this study was to determine whether the expression of the uPAR gene correlates with clinico-pathological parameters in human gastric carcinomas. Materials and Methods: We examined the expression of uPAR mRNA by using northern blot analysis and RT-PCR in 35 gastric carcinomas and the surrounding normal mucosa. Macroscopic and histopathological tumor findings and survival rates were obtained from the patient records and from endoscopic, surgical, and pathological reports. Results: The expression of uPAR and was higher in most neoplasms than in the corresponding normal mucosal tissue. uPAR mRNA expression in tumors correlated well with lymph-node metastasis (P<0.02) and tumor stage (P<0.01). The survival rate of patients with tumors displaying high uPAR expression levels was significantly lower (P<0.04) than that of patients without uPAR expression, but IL-8 showed only the tendency of survival difference. Conclusion: These results suggest that uPAR may be an important prognostic factor in human gastric carcinomas.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.1
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• pp.19-31
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• 2007

Objective: Pathogenesis of the endometriosis is very complex and the etiology is still unclear. Our hypothesis is that there may be some difference in gene expression patterns between eutopic endometriums with or without endometriosis. In this study, we analyzed the difference of gene expression profile with cDNA microarray. Methods: Endometrial tissues were gathered from patients with endometriosis or other benign gynecologic diseases. cDNA microarray technique was applied to screen the different gene expression profiles from early and late secretory phase endometria of those two groups. Each three mRNA samples isolated from early and late secretory phase of endometrial tissues of control were pooled and used as master controls and labeled with Cy3-dUTP. Then the differences of gene expression pattern were screened by comparing eutopic endometria with endometriosis, which were labeled with Cy5-dUTP. Fluorescent labeled probes were hybridized on a microarray of 4,800 human genes. Results: Twelve genes were consistently over-expressed in the endometrium of endometriosis such as ATP synthase H transporting F1 (ATP5B), eukaryotic translation elongation factor 1, isocitrate dehydrogenase 1 (NADP+), mitochondrial ribosomal protein L3, ATP synthase H+ transporting (ATP5C1) and TNF alpha factor. Eleven genes were consistently down-regulated in the endometriosis samples. Many extracellular matrix protein genes (decorin, lumican, EGF-containing fibulin-like extracellular matrix protein 1, fibulin 5, and matrix Gla protein) and protease/protease inhibitors (serine proteinase inhibitor, matrix metalloproteinase 2, tissue inhibitor of metalloproteinase 1), and insulin like growth factor II associated protein were included. Expression patterns of selected eight genes from the cDNA microarray were confirmed by quantitative RT-PCR or real time RT-PCR. Conclusion: The result of this analysis supports the hypothesis that the endometrium from patients with endometriosis has distinct gene expression profile from control endometrium without endometriosis.

• Tuberculosis and Respiratory Diseases
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• v.61 no.3
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• pp.256-264
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• 2006

Background: An acute lung injury(ALI) is characterized by the recruitment, activation, and apoptosis of inflammatory cells, numerous products released by inflammatory cells such as reactive oxygen species, inflammatory mediators, and a variety of proteolytic enzymes. It was reported that bacterial infections in diabetics showed impaired PMN functions such as reduced PMN respiratory burst and decreased microbicidal activity in inflamed tissue. However, the effect of the proteinase - inhibitor (MMP-9 vs TIMP-1) in ALI in diabetics is unclear. This study evaluated the differences in the expression of MMP-9 and TIMP-1 after the stimulation of endotoxin in a rat model. Methods: Six-week-old male Sprague-Dawley rats were classified into normal, DM, LPS and DM+LPS groups. The peripheral blood, BAL fluids, and lung tissues were obtained from individual rats. The MMP-9 activity was measured by gelatin zymography and the TIMP-1 level was measured by Western blotting. Results: The total BAL cells of the DM-LPS groups were significantly lower than the LPS groups (p < 0.01). The MMP-9 activities in the serum were higher in the DM+LPS groups than in the other groups. The MMP-9 activities in the BAL fluids were significantly higher in the DM+LPS group than in the normal and diabetic rats (p < 0.05). TIMP-1 expressions in the BAL fluids were significantly lower in the DM+LPS group than other groups (p < 0.05). The ratio between MMP-9 and TIMP-1 in the BAL fluids was significantly higher in the DM+LPS groups (p < 0.05). Conclusion: In ALI in diabetics the higher MMP-9 activity and lower TIMP-1 level are believed to prolonged and intensify the course of inflammation.

• Parasites, Hosts and Diseases
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• v.34 no.4
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• pp.247-254
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• 1996

Differentiation of invasive strains of Entamoebn histolytica according to their pathogenicity has been a topic of long debate, but now the pathogenic species only is regarded as E. histolytica while the non-pathogenic species is E. dispar. The present study applied immunoblot to differentiale infections of the two species among microscopically- detected cyst-passers in Korea. The crude extract of 5. histolyticn separated in 5-20% gradient gels, revealed many fractions of 94. 81. 71, 50. 44, 38.5. 37.5, 29, 19. and 18 kDa when the cysteine proteinase inhibitor. E64, was supplemented. The serum IgG antibody of 3 proven E. histolytirc cases reacted loth the antigenic fractions of 117. 110. 99.68,66,60.54.52, 46. and 45 kDa. Sera of PCR confirmed 3 cases of E. disper reacted only to the 117 kDa fraction or the E. histolytica crude extract which was regarded as non specific. To the antitigen of monoxenic E. dispar. sera or E. dispar and E. histolytica cases showed the same immunoblot reactions. The serum IgG antibody reacted with several antigenic fractions of both E. histolytica and E. dispar. but IgM and IgE antibodies showed no reaction to either antigen. Sera of 24 symptomless amebic cyst-passers were screened with the E. histolytica alltigen; two were found to be infected by E. histolytica and 22 were by E. dispar. The present findings suggest that in Korea most of asymptomatic cyst passers of E. histolytica are carriers of E. dispar. Immunoblot using E. histolytica antigen is a good technique for the differentiation of E. histolytica and E. dispar infections.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.22 no.2
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• pp.123-132
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• 2000

$TGF-{\beta}_1$ is a potent chemotactic factor for inflammatory cells and fibroblasts. It also stimulates the celluar source and components of extracellular matrix and the production of proteinase inhibitors. Collectively, these biologic activities lead to the accumulation and stabilization of the nascent matrix, which is vital to infection control. The objective of this study is to investigate production of $TGF-{\beta}$ in vitro fibroblast culture in the presence of Staphylococcus enterotoxin B(SEB) and/or lipopolysaccharide(LPS) and to elucidate the role of $TGF-{\beta}_1$ which may be responsible for infection control. The fibroblasts were originated from gingiva and facial dermis in 26 year-old male patient. In the presence of LPS($0.01{\mu}g$, $0.1{\mu}g$, $1.0{\mu}g$), SEB($0.01{\mu}g$, $0.l{\mu}g$, $1.0{\mu}g$) respectively, $cells(5{\times}10^3ml)$ were cultivated in vitro. At 1, 3, and 5 days after incubation, cells were counted. Also, $cells(2.5{\times}10^5ml)$ were cultivated in EMEM with LPS(0.01, 0.1 and $1.0{\mu}g$), SEB(0.01, 0.1 and $1.0{\mu}g$) respectively and $LPS(0.1{\mu}g)$ and $SEB(0.1{\mu}g)$ in combination for 24, 48, and 72 hours respectively. Culture supernatants were harvested at 1, 2, and 3 days after incubation period and triplicate culture supernatants were pooled and $TGF-{\beta}_1$ was assayed in duplicate. The results were as follows. 1. In gingival fibroblast induced with SEB and LPS respectively or in combination, the suppression of cell Proliferation occurred very significantly since 3 days after incubation, compared with the control and the production of $TGF-{\beta}_1$ occurred very significantly at 1 day after incubation, compared with the control. 2. In facial dermal fibroblast induced with SEB and LPS respectively or in combination, the suppression of cell proliferation occurred very significantly at 1 day after incubation, compared with the control. In SEB exposure, the production of $TGF-{\beta}_1$ was decreased very significantly at 1 day after incubation, compared with the control. However, in LPS, SEB and LPS exposure, the production of $TGF-{\beta}_1$ was increased very significantly at 1 day after incubation, compared with the control. In conclusion, the concentration of bacterial toxins and the incubation period correlated with cell proliferation and production of $TGF-{\beta}_1$ very significantly. The gingival and facial dermal fibroblasts have different phenotype each other The orchestrated understanding of fibroblast proliferation and $TGF-{\beta}_1$ production play an important part in host defense against the bacterial Infection and may prevent tissue necrosis such as necrotizing fasciitis and life-threatening syndrome such as multiple organ failure.

• Applied Biological Chemistry
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• v.37 no.2
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• pp.65-71
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• 1994

Chungkook-Jang was produced by fermenting Bacillus licheniformis CN-115. The changes of chemical composition, enzyme activity, and amino acids during the fermentation were investigated. The proximate composition was shown irregular fluctuation phenomenon during the fermentation, but only the moisture tended some reducing during the fermentation just after steaming. The content of amino nitrogen was increased radically after the 36 hours of fermentation and became the highest level at 18.072 mg/g at the 60 hours of it. In accordance with the fermentation of Chungkook-Jang, pH got to the 8.39 at 60 hours with increasing, protease activity was increased according to the fermentation and acid and neutral protease activity was reduced after being reached at the highest activity at 48 hours. The most suitable pH was 6.5 and temperature was $35^{\circ}C$ for dissolution-activated of protein in the process of fermentation of Chungkook-Jang. The content of water soluble protein and the content of salt soluble protein were increased at continuously according to the fermentation time of Chungkook-Jang the largest quantity. The molecular weight of water soluble protein of Chungkook-Jang fermented for 48 hours was about 19,000. The amino acids of water soluble protein just after steaming were totally 16 kinds and proline was amino acid and them was in series by glutamic acid and serine in that ordered. The amino acids salt soluble protein, just after steaming were totally 16 kinds and was the largest quantity phenylalanine, glutamic acid and aspartic acid and aspartic acid in that order.

• Journal of Acupuncture Research
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• v.19 no.2
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• pp.51-64
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• 2002

We have compared(using the same series of experimental tissue samples) the levels of proteolytic enzyme activities and free radical-induced protein damage in synovial fluid from RA and CPH cases. Many protease types showed significantly increased (typically by a factor of approximately 2-3-fold) activity in RA, compared to normal rats. However, CPH significantly reduced the cytoplasmic enzyme activities of arginyl aminopeptidase, leucyl aminopeptidase, pyroglutamyl aminopeptidase, tripeptidyl aminopeptidase, and proline endopeptidase to almost about 1/10 each. For the Iysosomal proteases, synovial fluid samples from RA rats, CPH significantly reduced the enzyme activities of cathepsin B, dipeptidyl aminopeptidase I and dipeptidyl aminopeptidase II. In extracellular matrix degrading(collagenase, tissue elastase) and leukocyte as sociated proteases (leukocyte elastase, cathepsin G), CPH decreased these enzyme activities of collagenase, tissue elastase and leukocyte associated elastase in RA. In cytoplasmic and lysosomal protease activities in plasma from RA. CPH and normal plasma samples were not significantly different, suggesting that altered activity of plasma proteases (particularly those enzymes putatively involved in the immune response) is not a contributory factor in the pathogenesis of RA. In addition, the level of free radical induced damage to synovial fluid proteins was approximately twice that in RA, compared with CPH. CPH significantly decreased the level of ROS induced oxidative damage to synovial fluid proteins (quantified as protein carbonyl derivative). Therefore we conclude that both proteolytic enzymes and free radicals are likely to be of equal potential importance as damaging agents in the pathogenesis of inflammatory joint disease, and that the design of novel therapeutic strategies for patients with the latter disorder should include both protease inhibitory and free radical scavenging elements. In addition, the protease inhibitory element should be designed to inhibit the action of a broad range of protease mechanistic types (i.e. cysteine-, metallo- and serine- proteinases and peptidases). However, increased protein damage induced by ROS could not be rationalised in terms of compromised antioxidant total capacity, since the latter was not significantly altered in RA synovial fluid or plasma compared with CPH.

• Journal of Chest Surgery
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• v.30 no.7
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• pp.677-685
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• 1997

Thromboelastography(TEG) is the unique measure that gives rapid information about the whole clotting process. Simplifying the diagnosis of coagulopathy during operations, TEG can provide an adequate therapy for postoperative bleeding. Remarkable improvement in hemostasis after cardiopulmonary bypass(CPB) has been achieved by the treatment with proteinase inhibitor aprotinin, but the hemostatic mechanism of aprotinin during CPB is still unclear. This study was designed to evaluate the effects of aprotinin on coagulation system during CPB by using TEG. Forty patients who underwent CPB were divided into two groups: aprotinin(2u 106 kallikrein inhibition units, as a single dose into the cardiopulmonary bypass priming solution) treatment group(male 14, female 8, mean age=50.Byears) and no aprotinin treatment(control) group(male 10, female 8, mean age=53.4 years). TEG, activated clotting time, prothrombin time, activated partial thromboplastin time, platelet counts, fibrinogen an (ibrinogen degradation product(FDP) concentrations were checked before and after CPB(30 minutes after neutralization of heparin effect by protamine sulfate). There was no significant difference in other conventional coagulation tests of two groups except postcardiopulmonary bypass FDP concentration in control group, which was significantly increased compared to that in aprotinin group(p<0.05). In TEG variables of both groups, clot formation time(K) and alpha $angle(\alpha^{\circ})$ were significantly increased and decreased, respectively, after CPB(p<0.05), but fibrinolytic index(LYS60) was not changed during CPB. In aprotinin group, reaction time(R) was decreased significantly after CPB(p<0.05) but maximum amplitude(MA) was not changed(p>0.05). On the contrary, R was not changed markedly but MA was decreased significantly in control group after CPB(p<0.05). This result shows that main change in coagulation system during CPB is not hyperfibrinolysis but cecrease in clot strength by platelet dys unction, and the main effect of aprotinin during cardiopulmonary bypass is the maintenance of clot strength to the pre-CPB level by the preservation of platelet function.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.19 no.6
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• pp.547-557
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• 1986

The characteristics of the three alkaline proteinases, Enz. A, B and C, from the pyloric caeca of mackerel have been investigated. The optimum condition for the activity of the Enz. A, B and C was pH 9.4, 9.8 and 9.8 at $45^{\circ}C$ for $2\%$ casein solution, and was pH 9.2 10.2 and 9.8 at $45^{\circ}C$ for $5\%$ hemoglobin denatured by urea, respectively. Enz. A, B and C by heat treatment at $50^{\circ}C$ for 5 min were inactivated 90, 33 and $37\%$, respectively, over the original activity. The reaction rate of the three alkaline proteinases was constant to the reaction time to 40 min in the reaction condition of $2{\mu}g/ml$ of enzyme concentration and $2\%$ casein solution. The reaction rate equation and Km value against casein substrate determined by the method of Lineweaver and Burk were: Enz. A, Y=3.6X and $Km=5.0{\times}10^{-3}\%$; Enz. B, Y=6.0X and $Km=1.0{\times}10^{-3}\%$; Enz. C, Y=4.2X and $Km=3.6{\times}10^{-3}\%$. The three alkaline proteinases were inactivated by $Ag^+$ and $Hg^{2+}$, but activated by $Mn^{2+},\;Sn^{2+}\;and\;Pb^{2+}$, Enz. B and C were remarkably inhibited by the soybean trypsin inhibitor. Molecular weight of Enz. A, B and C determined by SDS-polyacrylamide gel electrophoresis and Sephadex G-100 gel filtration was in the range of $27,500{\pm}2,500,\;20,500{\pm}1,500\;and\;15,250{\pm}250$, respectively.