• Journal of Microbiology and Biotechnology
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• v.32 no.11
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• pp.1462-1470
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• 2022

Natural antimicrobial substances are needed as alternatives to synthetic antimicrobials to protect against foodborne pathogens. In this study, a bacteriocin-producing bacterium, Bacillus subtilis HD15, was isolated from doenjang, a traditional Korean fermented soybean paste. We sequenced the complete genome of B. subtilis HD15. This genome size was 4,173,431 bp with a G + C content of of 43.58%, 4,305 genes, and 4,222 protein-coding genes with predicted functions, including a subtilosin A gene cluster. The bacteriocin was purified by ammonium sulfate precipitation, Diethylaminoethanol-Sepharose chromatography, and Sephacryl gel filtration, with 12.4-fold purification and 26.2% yield, respectively. The purified protein had a molecular weight of 3.6 kDa. The N-terminal amino acid sequence showed the highest similarity to Bacillus subtilis 168 subtilosin A (78%) but only 68% similarity to B. tequilensis subtilosin proteins, indicating that the antimicrobial substance isolated from B. subtilis HD15 is a novel bacteriocin related to subtilosin A. The purified protein from B. subtilis HD15 exhibited high antimicrobial activity against Listeria monocytogenes and Bacillus cereus. It showed stable activity in the range 0-70℃ and pH 2-10 and was completely inhibited by protease, proteinase K, and pronase E treatment, suggesting that it is a proteinaceous substance. These findings support the potential industrial applications of the novel bacteriocin purified from B. subtilis HD15.

• Journal of Animal Science and Technology
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• v.49 no.5
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• pp.559-568
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• 2007

Recently, copy number variations (CNV) of genes or genomic segments have been intensively studied and various analysis methods have been developed. In this study, quantitative oligonucleotide ligation assay (qOLA) was applied to investigate CNV of KIT gene in the Landrace breed. A combined assay using qOLA and pyrosequencing, 6 genotype classes, I1/I1 or I3/i (IBe), I1/I2 or I3/IP, I1/I3, I1/IP or I2/i (IBe), I2/I2and I2/IP, were identified from 44 Landrace pigs. Genotype assignment using grouping features of measurements on a scatter plot showed 100% agreement with those using a statistical assignment by PROC FASTCLUS procedure implemented in the SAS package. Two versions (3100 and 3130) of ABI sequencers gave the same genotyping results, indicating there was no influence on qOLA by different versions of instrument, however, the means of standard deviation and coefficient of variation from the qOLA on a ABI 3130 (2.33 and 4.10) was lower than those from the qOLA on a ABI 3100 (2.67 and 4.81). Effect of proteinase K treatment on the PCR product followed by qOLA was very clear because noise peaks were disappeared and the observed ration fit better to the reference ratio corresponding to each genotype.

• Journal of the Korean Society of Food Science and Nutrition
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• v.40 no.6
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• pp.903-911
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• 2011

In this study, Bacillus polyfermenticus CJ6 harboring antibacterial activity was isolated from meju. The antibacterial activity of Bacillus polyfermenticus CJ6 was stable in the pH range of 3.0~9.0, but it disappeared after culture at $70^{\circ}C$ for 24 hr. Antibacterial activity was inactivated by proteinase K, protease, and ${\alpha}$-chymotrypsin, indicating its proteinaceous nature. The growth inhibitory effects of B. polyfermenticus CJ6 culture on food-borne pathogens such as Staphylococcus aureus, Salmonella Typhi, Listeria monocytogenes, and Escherichia coli O157:H7 were examined in this study. Approximately 6~6.2 log CFU/mL of each pathogen was co-cultured with B. polyfermenticus CJ6 in a 50 mL culture volume for 24 hr. Growth of S. aureus and L. monocytogenes was completely inhibited after 3 hr of incubation. Growth of S. Typhi and E. coli O157:H7 was also completely inhibited after 6 hr of incubation. The antibacterial compounds from B. polyfermenticus CJ6 were purified by solid phase extraction (C18 Sep-pak cartridge), recycling preparative HPLC, and analytical HPLC. Ultra-high performance liquid chromatography and electrospray ionization tandem mass spectrometry analysis were used to identify the purified antibacterial compounds, which were confirmed to be five peptides (757.4153 Da, 750.3444 Da, 1024.5282 Da, 1123.6083 Da, and 1617.8170 Da).

• Journal of Animal Science and Technology
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• v.48 no.6
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• pp.907-920
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• 2006

This study was conducted to investigate the inhibitory activity of Lactobacillus spp., Bacillus ssp., and calf fecal isolates against pathogenic Salmonella typhimurium, E. coli, Listeria monocytogenes, and Staphylococcus aureus. Among thirteen strains of Lactobacillus ssp. tested, Lactobacillus helveticus CU631 showed the highest inhibition against three pathogens, whereas Bacillus spp. showed a weak inhibitory activity. Four calf fecal isolates were identified as Lactobacillus pentosus CU13, CU05, Pediococcus pentosaceus CUR02, and Lactobacillus lactis ssp. lactis CUM14. The whole cell and cell wall components of L. rhamnosus CU02 and L. pentosus CU13 were active in the inhibition of L. monocytogenes. The medium components and levels, which affect on the inhibitory activity, were revealed as Tween 80 1.0%, peptone 3.0%, yeast extract 3.0%, glucose 3.0%, beef extract 3.0%, and NaCl 1.0～3.0%, respectively. Inhibitory activity of the supernatant culture medium was not affected by catalase and proteinase K treatment but affected by heat treatment at 80℃ and netralization, which implies that the inhibitory activity is due to the production of organic acids during the growth. L. pentosus CU13 and L. rhamnosus CU02 exhibited broad inhibition spectrum against 16 out of 21 strains including some pathogens. Oral administration of L. rhamnosus CU02 to the mice infected with E. coli O157:H7 was proven to be effective to recover their body weight during the experimental period.

• Microbiology and Biotechnology Letters
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• v.7 no.2
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• pp.75-89
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• 1979

One strain of Penicillium sp. (175-66-B), isolated from soil, was able to produce a substance that has a strong inibition activity against the Agkistrodon and Trimeresurus venoms. In this experiment, the chemical and biological properties of the sample were investigated. As an inhibitory substance, it was effective to the proteinase, hemorrhagic and lethal factors of Agkistrodon and Trimeresurus venoms, and also effective to several fractions of the proteinases and hemorrhagic factors of Agkistrodon halys blomhoffi venom. Moreover, in the addition of prednisotone, it was more effective for the cure of the mouse envenomated with the venom amount of two fold of MLD$_{100}$. This substance was very stable to the acid, alkali and heat. Its melting point was high enough to sublime at 222$^{\circ}C$ without any decomposition. This sample was easily dissolved only in hot water, but not in several organic solvents except for a little dissolution in elate. It did not have the chelating activity. It had very strong specificity to the snake venoms. but its activity was depressed by the addition of zinc or cupric salts. This sample had no acute toxicity to the mouse. Its chemical formula was $C_{16}$ $H_{12}$$N_2$ $O_{10}$ with the molecular weight of about 392. It has two epoxy groups and four carboxyl radicals, but amino, nitrite and nitrate radicals, unsaturated bonds and aromatic ring were not detected. Theuchemical configuration of this sample was suggested to be;

• Parasites, Hosts and Diseases
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• v.36 no.4
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• pp.255-260
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• 1998

To evaluate the biological and biochemical characteristics of Trichomonas vaginalis KT9 isolate, the growth and size of trichomonads, pathogenicity in mouse, protein profiles and proteinase activity were examined after shifting the medium from TPS-1 into TYM. Generation time of trichomonads in TYM medium was 4.5 hr in comparison to TPS-1 with 7.1 hr. Size of trichomonads cultured in TPS-1 medium ($8.5{\;}{\pm}{\;}0.9{\;}{\times}{\;}6.0{\;}{\pm}{\;}0.9{\;}{\mu\textrm{m}}$) was significantly smaller than those in TYM medium ($10.9{\;}{\pm}{\;}1.4{\;}{\times}{\;}8.2{\;}{\pm}{\;}0.9{\;}{\mu\textrm{m}}$). Trichomonads cultured in TYM medium produced subcutaneous abscess in 9 out of 10 mice, whereas those in TPS-1 medium produced abscesses in 2 out of 10 mice. In SDS-PAGE, trichomonad Iysates from both media showed ten common bands. However, trichomonads in TYM medium showed additional bands of 136 kDa, 116 kDa and 40 kDa in comparison to those in TPS-1 with 100 kDa. By immunoblot with T. vaginalis-immunized rabbit sera, T. vaginalis cultivated in both TYM and TPS-1 media showed 5 common bands. and unique bands of 116 kDa. 105 kDa. and 86 kDa were observed in trichomonads in TYM while a 140 kDa band in those in TPS-1. In gelatin SDS-PAGE, trichomonads in TYM degraded gelatin stronger than those in TPS-1. Also protease activity of trichomonads in TYM was significantly higher than that of trichomonads in TPS-1 using Bz-Pro-Phe-Arg-Nan as a substrate. According to the results, it is assumed that the shift from TPS-1 into TYM medium for cultivation of T. vaginalis might modulate the biological and biochemical properties of T vaginalis in vitro.

• Radiation Oncology Journal
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• v.26 no.1
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• pp.56-64
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• 2008

Purpose: Cathepsin D(CD) is a lysosomal acid proteinase that is related to malignant progression, invasion, and a poor prognosis in several tumors. The aim of this study was to evaluate the prognostic clinical significance of CD and p53 expression in pretreatment biopsy specimens from patients with locally advanced rectal cancer who were treated with preoperative chemoradiation. Materials and Methods: Eighty-nine patients with locally advanced rectal cancer(cT3/T4 or N+) were included in this study. Preoperative chemoradiation consisted of a dose of 50.4 Gy of pelvic radiation and two concurrent cycles of administration of 5-fluorouracil and leucovorin. Surgery was performed six weeks after chemoradiation. CD and p53 expression in pretreatment formalin-fixed paraffin-embedded tumor biopsy specimens were assessed by immunohistochemical staining using a CD and p53 monoclonal antibodies. The threshold value for a positive stain in tumor tissue and stromal cells was 1+ intensity in 10% of the tumors or stromal cells, respectively. Results: Positive CD expression was found in 57(64%) of the tumors and 32(35%) of the stromal cell specimens. There was no association with CD expression of the tumor or stromal cells and patient characteristics. There was a correlation between tumor CD expression with stromal cell CD expression(p=0.01). Overexpression of p53 was not a significant prognostic factor. The 5-year overall survival(OS) and disease-free survival(DFS) rates were not different between tumor CD-negative and positive patient biopsy samples(69% vs. 65%, 60% vs. 61%, respectively). The 5-year OS rates in the tumor-negative/stromal cell-negative, tumor-negative/stromal cell-positive, tumor-positive/stromal cell-negative and tumor-positive/stromal cell-positive biopsy samples were 75%, 28%, 62%, and 73%, respectively. Stromal cell staining only without positive tumor staining demonstrated the worst overall survival prognosis for patients(p=0.013). Conclusion: Overexpression of p53 in rectal biopy tissue was not associated with prognostic significance. In the pretreatment biopsy specimens, an exclusive increase in CD expression in stromal cells without tumor expression was related to poor overall survival in patients with locally advanced rectal cancer treated with preoperative chemoradiation.

• Childhood Kidney Diseases
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• v.12 no.2
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• pp.164-169
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• 2008

Purpose : There have been some reports on the prevalence of positive antineutrophil cytoplasmic antibody(ANCA) in Henoch-$Sch{\ddot{o}}nlein$ purpura(HSP), but the results were conflicting. We performed this study to evaluate the clinical significance of ANCA(c-ANCA and p-ANCA) in Korean children with HSP. Methods : The medical records of 30 patients(13 boys and 17 girls) aged 6.0$\pm$1.9(5-12) years with a clinical diagnosis of HSP based on the EULAR/PReS criteria were reviewed retrospectively. From the years 2007 to 2008, the sera from children with acute HSP were tested for antineutrophil cytoplasmic antibodies(ANCA). The target antigens of these autoantibodies are proteinase 3(c-ANCA) or myeloperoxidase(p-ANCA). Results : Palpable purpura was seen in all 30 patients(100%), abdominal pain in 20(67%), arthralgia in 17(57%), and renal involvement in 11(37%). Laboratory findings showed leukocytosis in 4 patients(13%), thrombocytosis 18 in(60%), and elevated erythrocyte sedimentation rate in 18(60%). Anti-streptolysin O titers were elevated in 7% of the patients and no patient showed elevation of serum IgA level. The sera from 29 patients were negative for c-ANCA and p-ANCA by indirect immunofluorescence, but only one patient had weakly positive results, which became negative at follow-up. Conclusions : We conclude that c-ANCA or p-ANCA is not an important serologic marker in children with HSP, because it was neither diagnostically nor immunologically specific in children with HSP. These results suggest that ANCA are not involved in the pathogenesis of HSP in children.

• Development and Reproduction
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• v.5 no.1
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• pp.23-33
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• 2001

When mammalian oocytes ovulate into the oviduct, associating follicular fluid components are exposed to the oviductal environment, possibly resulting in the mutual interaction between fillicu1ar and oviductal fluids. In the Present study, we have demonstrated for the first time that components of fallicular fluid could be modified by the oviductal fluid. Gelatin zymographic analyses of human follicular fluid (hFF) obtained from IVF patients showed consistently the presence of 110 kDa gelatinase (GA110) in addition to many bands among which 62 kDa gelatinase was predominant. Addition of EDTA or phenanfhroline to the gelatinase substrate buffer during gel incubation abolished GA110 band whereas phenylmethylsulffnyl fluoride (PMSF) did not. In contrast, bovine oviductal fluid(bOF) exhibited only 62 kDa gelatinase. Surprisingly, when bOF was added to hFF in 1:1 ratio and then the mixture was incubated for 3 h at 37$^{\circ}$C, GA110 of hFF disappeared. Disappearance of GA110 by bOF was observed even within 30 min after mixing with hFF. Addition of aminophenylmercuric acetate (APMA) to hFF also abolished enzymatic activity of GA110 but increased the activityof 62 kDa gelatinase. However, APMA abolished many other gelatinases as well unlike bOF. Interestingly, treatment of hFF with EDTA for 3 h remarkably increased the enzymatic activity of GA110 but not that of other gelatinases. Addition of phenanthroline, PMSF or soybean trypsin inhibitor (SBTI) did not affect overall gelatinase activities. Again, addition of bOF to the hFF pretreated with any of the above proteinase inhibitors abolished the appearance of GA110. Human serum also showed GAI 10 of which activity was greatlyenhanced by EDTA treatment. Similar to hFF, serum GA110 also disappeared by the addition of bOF. Human granulosa cell homogenate did not reveal any appreciable gelatinase activity except 92 kDa gelatinase. Anti-human gelatinase A antibody reacted with 62 kDa gelatinase of hFF. Based upon these results, it is concluded that bOF could selectively degrade an isoform of gelatinase A present in hFF and human serum.