• Restorative Dentistry and Endodontics
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• v.40 no.4
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• pp.306-313
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• 2015

Objectives: Molecular mechanism of the pathogenicity of Enterococcus faecalis (E. faecalis), a suspected endodontic pathogen, has not yet been adequately elucidated due to limited information on its virulence factors. Here we report the identification of in vivo expressed antigens of E. faecalis by using a novel immunoscreening technique called change-mediated antigen technology (CMAT) and an experimental animal model of endodontic infection. Materials and Methods: Among 4,500 E. coli recombinant clones screened, 19 positive clones reacted reproducibly with hyperimmune sera obtained from rabbits immunized with E. faecalis cells isolated from an experimental endodontic infection. DNA sequences from 16 of these in vivo-induced (IVI) genes were determined. Results: Identified protein antigens of E. faecalis included enzymes involved in housekeeping functions, copper resistance protein, putative outer membrane proteins, and proteins of unknown function. Conclusions: In vivo expressed antigens of E. faecalis could be identified by using a novel immune-screening technique CMAT and an experimental animal model of endodontic infection. Detailed analysis of these IVI genes will lead to a better understanding of the molecular mechanisms involved in the endodontic infection of E. faecalis.

• BMB Reports
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• v.52 no.12
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• pp.712-717
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• 2019

Translocase of outer mitochondrial membrane 20 (TOMM20) plays an essential role as a receptor for proteins targeted to mitochondria. TOMM20 was shown to be overexpressed in various cancers. However, the oncological function and therapeutic potential for TOMM20 in cancer remains largely unexplored. The purpose of this study was to elucidate the underlying molecular mechanism of TOMM20's contribution to tumorigenesis and to explore the possibility of its therapeutic potential using colorectal cancer as a model. The results show that TOMM20 overexpression resulted in an increase in cell proliferation, migration, and invasion of colorectal cancer (CRC) cells, while siRNA-mediated inhibition of TOMM20 resulted in significant decreases in cell proliferation, migration, and invasion. TOMM20 expression directly impacted the mitochondrial function including ATP production and maintenance of membrane potential, which contributed to tumorigenic cellular activities including regulation of S phase cell cycle and apoptosis. TOMM20 was overexpressed in CRC compared to the normal tissues and increased expression of TOMM20 to be associated with malignant characteristics including a higher number of lymph nodes and perineural invasion in CRC. Notably, knockdown of TOMM20 in the xenograft mouse model resulted in a significant reduction of tumor growth. This is the first report demonstrating a relationship between TOMM20 and tumorigenesis in colorectal cancer and providing promising evidence for the potential for TOMM20 to serve as a new therapeutic target of colorectal cancer.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2095-2105
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• 2018

In our previous studies, we have identified several in vivo-induced antigens and evaluated their potential as subunit vaccine candidates in a murine model, in which the recombinant protein GalT showed the most potent immunogenicity and immunoprotective efficacy against Actinobacillus pleuropneumoniae. To exploit a more efficient way of delivering GalT proteins, in this study, we employed the widely studied E. coli outer membrane vesicles (OMVs) as a platform to deliver GalT protein and performed the vaccine trial using the recombinant GalT-OMVs in the murine model. Results revealed that GalT-OMVs could elicit a highly-specific, IgG antibody titer that was comparable with the adjuvant GalT group. Significantly higher lymphocyte proliferation and cytokines secretion levels were observed in the GalT-OMVs group. 87.5% and 50% of mice were protected from a lethal dose challenge using A. pleuropneumoniae in active or passive immunization, respectively. Histopathologic and immunohistochemical analyses showed remarkably reduced pathological changes and infiltration of neutrophils in the lungs of mice immunized with GalT-OMVs after the challenge. Taken together, these findings confirm that OMVs can be used as a platform to deliver GalT protein and enhance its immunogenicity to induce both humoral and cellular immune responses in mice.

• Korean Journal of Poultry Science
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• v.48 no.4
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• pp.327-335
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• 2021

Riemerella anatipestifer (RA) can cause septicemia, polyserositis, and ataxia in ducks. It can also colonize the upper respiratory tract of healthy ducks. These differences in pathogenicity are probably the result of diverse mechanisms of virulence in different strains. Since serum resistance is a feature frequently found in systemic pathogens, 130 RA strains having different clinical origins were tested. A variety of serum susceptibility levels were detected. Pharynx strains from healthy ducks were mainly susceptible to the bactericidal effect of the serum, while systemic strains were serum resistant. Heat-treatment of the sera abolished the bactericidal activity, indicating that complement is a key factor in this effect. In an attempt to associate serum-resistance to surface determinant genes of the bacteria, we screened for six genes involved in lipopolysaccharide synthesis and membrane proteins in RA. Of these, three genes (AS87_09335, AS87_00480, and AS87_05195) encoding outer membrane proteins might be implicated in serum resistance statistically. The results indicate that serum resistance is a virulence mechanism in RA.

• Applied Microscopy
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• v.27 no.3
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• pp.225-234
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• 1997

Vincristine, a kind of anticancerous drugs, interferes with development of microtubles and synthesis of nucleic acid and proteins in cells, and destructs cytoplasmic membrane so that mitosis of cancer cells is inhibited. Unfortunately these anticancerous effects by vioneristime are not limited to specific cancer cells, so several side effects are produced. This study was performed to explore the effects of vincristine on the fine structure of cytoplasmic organelles and cartilagenous matrix in proximal epiphyseal plate of the tibia in rat. The results were as follows: 1. Cisternae of rough endoplasmic reticulum (RER) were fragmented and sacculated, and membrane-bound ribocomes of RER were detached at 3 and 6 hours after vincristine treatment. Severely dilated, fagmented and sacculated cisternae of RER were found at 12 hours after vincristine treatment, and at 24 hours after vincristine treatment a few cisternae were framented and sacculated. At 72 hours after vincristine treatment cisternae of RER were parallely well arraged. 2. Golgi complex was atrophied at 3, 6, and 12 hours after vincristine treatment, while at 72 hours after vincristine treatment the cisternae of Golgi complex were made of 5-6 layers. 3. Mitochondria with disorganized mitochondrial cristae and outer membrane-losed mitochondria were found at 3 hours after vincristine treatment. At 6 and 12 hours after vincristine treatment mitochondria had possessed disorganized cristae, and a few mitochondria with disorganized cristae were. observed at 24 hours after vincristine treatment. While at 72 hours after vincristine treatment mitochondria were shown distinct cristae and double membranes. 4. Phagosome were begun to observe at 3 hourse after vincristine treatment, and at 24 hourse after vincristine treatment many phagosomes were found, while at 72 hours after vincristine treatment a few phagosomes were observed. 5. In the cartilagenous matrix large-sized matrix granules were decreased and collagen fibrils were dispersed at 3, 6, and 12 hours after vincristine treatment, while at 72 hours after vincristine treatment many large-sized matrix granules and numerous matrix it is suggested that although vincristine may induce the degenerative changes of the chondrocyte, resulting in changes of components of the cartilagenous matirx, these toxic effects may be regressed with time.

• Journal of Microbiology and Biotechnology
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• v.28 no.1
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• pp.157-164
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• 2018

Francisella tularensis (FT), a highly infectious pathogen, is considered to be a potential biological weapon owing to the current lack of a human vaccine against it. Tul4 and FopA, both outer membrane proteins of FT, play an important role in the bacterium's immunogenicity. In the present study, we evaluated the immune response of mice - humanized with human CD34+ cells (hu-mice) - to a cocktail of recombinant Tul4 and FopA (rTul4 and rFopA), which were codon-optimized and expressed in Escherichia coli. Not only did the cocktail-immunized hu-mice produce a significant human immunoglobulin response, they also exhibited prolonged survival against an attenuated live vaccine strain as well as human T cells in the spleen. These results suggest that the cocktail of rTul4 and rFopA had successfully induced an immune response in the hu-mice, demonstrating the potential of this mouse model for use in the evaluation of FT vaccine candidates.

• Journal of Microbiology and Biotechnology
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• v.26 no.9
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• pp.1613-1619
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• 2016

Francisella tularensis is a highly virulent pathogen of humans and other mammals. Moreover, F. tularensis has been designated a category A biothreat agent, and there is growing interest in the development of a protective vaccine. In the present study, we determine the in vitro and in vivo immune responses of a subunit vaccine composed of recombinant peptides Tul4 and FopA from epitopes of the F. tularensis outer membrane proteins. The recombinant peptides with adjuvant CpG induced robust immunophenotypic change of dendritic cell (DC) maturation and secretion of inflammatory cytokines (IL-6, IL-12). In addition, the matured DCs enabled ex vivo proliferation of naive splenocytes in a mixed lymphocyte reaction. Lastly, we determined the in vivo immune response by assessment of antibody production in C57BL/6 mice. Total IgG levels were produced after immunization and peaked in 6 weeks, and moreover, Tul4-specific IgG was confirmed in the mice receiving peptides with or without CpG. Based on these results, we concluded that the recombinant peptides Tul4 and FopA have immunogenicity and could be a safe subunit vaccine candidate approach against F. tularensis.

• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1476-1484
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• 2015

Three endoxylanase-encoding genes from the moderately themophilic chemoorganotrophic bacterium Melioribacter roseus were cloned and expressed in Escherichia coli. Genes xyl2091 (Mros_2091) and xyl2495 (Mros_2495) encode GH10 family hydrolases, whereas xyl2090 (Mros_2090) represents the GH30 family. In addition to catalytic domains, Xyl2090 and Xyl2091 contain carbohydrate-binding modules that could facilitate their binding to xylans and Por sorting domains associated with the sorting of proteins from the periplasm to the outer membrane, where they are covalently attached. Recombinant endoxylanase Xyl2495 exhibited a high specific activity of 1,920 U/mg on birchwood xylan at 40℃. It is active at low temperatures, exhibiting more than 30% of the maximal activity even at 0℃. Endoxylanases Xyl2090 and Xyl2091 have lower specific activities but higher temperature optima at 80℃ and 65℃, respectively. Analysis of xylan hydrolysis products revealed that Xyl2090 generates xylo-oligosaccharides longer than xylopentaose. Xylose and xylobiose are the major products of xylan hydrolysis by the recombinant Xyl2091 and Xyl2495. No activity against cellulose was observed for all enzymes. The presence of three xylanases ensures efficient xylan hydrolysis by M. roseus. The highly processive "free" endoxylanase Xyl2495 could hydrolyze xylan under moderate temperatures. Xylan hydrolysis at elevated temperatures could be accomplished by concerted action of two cell-bound xylanases; Xyl2090 that probably degrades xylans to long xylo-oligosaccharides, and Xyl2091 hydrolyzing them to xylose and xylobiose. The new endoxylanases could be useful for saccharification of lignocellulosic biomass in biofuels production, bleaching of paper pulp, and obtaining low molecular weight xylooligosaccharides.

• Microbiology and Biotechnology Letters
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• v.41 no.2
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• pp.242-248
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• 2013

Endotoxins are part of the outer membrane of the cell wall of gram-negative bacteria and are continuously released during bacterial growth. Endotoxins typically induce severe sepsis and septic shock, which cause more than 50% of mortalities. Endotoxins are easily measured in the serum by the limulus amebocyte lysate (LAL) test. However, a nonspecific result is obtained, because the high concentration of serum proteins disturbs the enzyme reaction of the LAL test. In order to solve this problem, the LAL test was performed in this study after the centrifugation of the boiled serum samples to remove the impurities. As a result, among the various conditions examined, endotoxin measurement with the LAL test was the most accurate and repeatable after centrifugation of the boiled serum at $100^{\circ}C$. Moreover, the endotoxin was accurately and repeatedly measured from the prepared sera of mice that had been administered an intraperitoneal injection of purified lipopolysaccharides (LPS) or E. coli. Therefore, the application of centrifugation to remove impurities from boiled serum gives an accurate measurement of endotoxins in the sera of normal subjects or patients, and this will lead to the improved diagnosis and prevention of diseases caused by endotoxins. In addition, the centrifugation of boiled serum samples should be considered and included in the development of endotoxin test kits.

• Journal of Microbiology and Biotechnology
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• v.30 no.4
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• pp.497-504
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• 2020

For control of brucellosis in small ruminants, attenuated B. melitensis Rev1 is used but it can be virulent for animals and human. Based on these aspects, it is essential to identify potential immunogens to avoid these problems in prevention of brucellosis. The majority of OMPs in the Omp25/31 family have been studied because these proteins are relevant in maintaining the integrity of the outer membrane but their implication in the virulence of the different species of this genus is not clearly described. Therefore, in this work we studied the role of Omp31 on virulence by determining the residual virulence and detecting lesions in spleen and testis of mice inoculated with the B. melitensis LVM31 mutant strain. In addition, we evaluated the conferred protection in mice immunized with the mutant strain against the challenge with the B. melitensis Bm133 virulent strain. Our results showed that the mutation of omp31 caused a decrease in splenic colonization without generating apparent lesions or histopathological changes apparent in both organs in comparison with the control strains and that the mutant strain conferred similar protection as the B. melitensis Rev1 vaccine strain against the challenge with B. melitensis Bm133 virulent strain. These results allow us to conclude that Omp31 plays an important role on the virulence of B. melitensis in the murine model, and due to the attenuation shown by the strain, it could be considered a vaccine candidate for the prevention of goat brucellosis.