• Journal of Microbiology and Biotechnology
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• 제30권10호
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• pp.1583-1591
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• 2020

CRISPR/Cpf1 has emerged as a new CRISPR-based genome editing tool because, in comparison with CRIPSR/Cas9, it has a different T-rich PAM sequence to expand the target DNA sequence. Single-base editing in the microbial genome can be facilitated by oligonucleotide-directed mutagenesis (ODM) followed by negative selection with the CRISPR/Cpf1 system. However, single point mutations aided by Cpf1 negative selection have been rarely reported in Corynebacterium glutamicum. This study aimed to introduce an amber stop codon in crtEb encoding lycopene hydratase, through ODM and Cpf1-mediated negative selection; deficiency of this enzyme causes pink coloration due to lycopene accumulation in C. glutamicum. Consequently, on using double-, triple-, and quadruple-base-mutagenic oligonucleotides, 91.5-95.3% pink cells were obtained among the total live C. glutamicum cells. However, among the negatively selected live cells, 0.6% pink cells were obtained using single-base-mutagenic oligonucleotides, indicating that very few single-base mutations were introduced, possibly owing to mismatch tolerance. This led to the consideration of various target-mismatched crRNAs to prevent the death of single-base-edited cells. Consequently, we obtained 99.7% pink colonies after CRISPR/Cpf1-mediated negative selection using an appropriate single-mismatched crRNA. Furthermore, Sanger sequencing revealed that single-base mutations were successfully edited in the 99.7% of pink cells, while only two of nine among 0.6% of pink cells were correctly edited. The results indicate that the target-mismatched Cpf1 negative selection can assist in efficient and accurate single-base genome editing methods in C. glutamicum.

• International Journal of Oral Biology
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• 제33권3호
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• pp.77-82
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• 2008

Innate immune response is initiated by the recognition of unique microbial molecular patterns through pattern recognition receptors (PRRs). The purpose of this study is to dissect the expression of various PRRs in gingival epithelial cells of differentiated versus undifferentiated states. Differentiation of immortalized human gingival epithelial HOK-16B cells was induced by culture in the presence of high $Ca^{2+}$ at increased cell density. The expression levels of various PRRs in HOK-16B cells were examined by realtime reverse transcription polymerase chain reaction (RTPCR) and flow cytometry. In addition, the expression of human beta defensins (HBDs) was examined by real time RT-PCR and the amounts of secreted cytokines were measured by enzyme linked immunosorbent assay. In undifferentiated HOK-16B cells, NACHT-LRR-PYDcontaining protein (NALP) 2 was expressed most abundantly, and toll like receptor (TLR) 2, TLR4, nucleotide-binding oligomerization domain (NOD) 1, and NOD2 were expressed in substantial levels. However, TLR3, TLR7, TLR8, TLR9, ICE protease-activating factor (IPAF), and NALP6 were hardly expressed. In differentiated cells, the levels of NOD2, NALP2, and TLR4 were different from those in undifferentiated cells at RNA but not at protein levels. Interestingly, differentiated cells expressed the increased levels of HBD-1 and -3 but secreted reduced amount of IL-8. In conclusion, the repertoire of PRRs expressed by gingival epithelial cells is limited, and undifferentiated and differentiated cells express similar levels of PRRs.

• KSBB Journal
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• 제16권6호
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• pp.527-532
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• 2001

공업용 유기용매나 그리스 제거제, 전자기판 세척제로 널리 사용되어 온 TCE는 적절한 처리 방법이 개발되지 못하여 환경오염을 일으키고 있으며, 유전자변이 및 암을 유발하는 것으로도 알려져 효율적인 처리기술 개발이 요구된다. 미생물막을 이용한 처리 방법은 TCE를 무해한 최종산물로 분해시킬 수 있으며, 운전비 및 설비비도 낮아 경쟁력 있는 처리 기술로 평가되고 있다. TCE 자체는 미생물의 성장기질로 사용되지 못하므로 공동대사 과정을 거쳐 분해된다. TCE 및 TCE 분해산물의 높은 독성으로 인하여 TCE 분해효소 및 세포 분해산물의 높은 독성으로 인하여 TCE 분해효소 및 세포 불활성화가 일어나서 장기간 안정된 반응기 운전이 어렵다는 단점과 성장기질을 공급하는 경우 유발되는 경쟁저해로 인한 분해효율 감소를 막을 수 있는 여러 종류의 생물반응기 시스템들이 개발되었다. 이러한 생물반응기 시스템의 개발과 우수한 분해능을 가지는 균주 선별 및 개량 등을 통해 실제 현장에 적용시킬 수 있는 생물학적 TCE 처리 기술의 실용화가 가능할 것이다.

• 식물조직배양학회지
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• 제21권6호
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• pp.357-362
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• 1994

키틴 가수분해 효소와 베타-1,3-글루카네이즈는 식물체의 주요 방어효소로 여겨지고 있다. 본 논문에서는 아기장대풀의 잎조직으로부터 고정된 현탁배양주를 만들고 이어서 에틸렌과 유인제 (elicitor) 처리에 의한 키틴 가수분해 효소유도양상을 분석하였다. 키틴 가수분해 효소활성은 $^3$H으로 표지된 키토산을 기질로 한 radiochemical 분석방법이나 담배식물체의 키틴 가수분해 효소를 대상으로 얻어진 항체를 이용하여 Western 분석방법을 사용하였다. 이 결과 에틸렌과 유인제 처리 공히 48시간후에 가장 활성이 높게 나타남을 관찰하였다. 또한 씨앗, 새싹, 식물체의 잎, 뿌리 등에서의 활성을 조사하여 뿌리에서 키틴 가수분해 효소 활성이 높음을 확인하였다.

• 한국지하수토양환경학회지:지하수토양환경
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• 제22권2호
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• pp.41-51
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• 2017

Microbial processes that bind heavy metals and form minerals are widespread, and they represent a basic aspect of biogeochemistry. Some microorganisms can crystallize minerals by secreting a specific enzyme. In particular, calcite ($CaCO_3$) precipitation is an important part of biomineralization, and has been studied extensively because of its wide application in civil engineering technology. This process provides an effective way to stabilize heavy metals within a relatively stable crystal phase. In this study, biomineralization of calcite by three urea-hydrolyzing indigenous bacterial strains was investigated by microbiological analyses. Three bacterial strains were isolated from the sludge of B mine in Mexico and each bacterial strain was identified by the cellular fatty acid composition and 16S rRNA partial sequencing analysis. The results of the identification analysis showed that these strains were closest to Sporosarcina pasteurii, Kurthia gibsonii, and Paenibacillus polymyxa. We found that the optimum conditions for growth of these indigenous bacteria were $30-40^{\circ}C$ and pH range of 7-8. Microbiological analyses showed the possibility that the bioaccumulated heavy metals ions were deposited around the cell as crystalline carbonate minerals under the optimum conditions. The findings of our study suggest that the indigenous bacterial strains play an important role in heavy metal immobilization.

• Asian-Australasian Journal of Animal Sciences
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• 제16권8호
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• pp.1158-1164
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• 2003

In two feeding experiments male and mixed-sex broiler chicks were offered diets based on sorghum and a wheatsorghum blend with two tiers of nutrient specifications, without and with microbial phytase (600 and 800 FTU/kg), from 7-25 and 1-42 days post-hatch, respectively. The nutrient specifications for protein, amino acids, energy density and phosphorus (P) of standard diets were reduced to formulate the modified diets on a least-cost basis. Calculated differences in nutrient specifications between standard and modified diets ranged from 14.3 to 17.1 g/kg crude protein, 0.24 to 0.40 MJ/kg apparent metabolisable energy (AME) and 1.06 to 1.20 g/kg available P. In both experiments, reduced nutrient specifications had a negative impact on growth rates and feed efficiency and phytase supplementation had a positive influence on growth performance and protein efficiency ratios (PER). Phytase addition to the less expensive, modified diets either partially or entirely compensated for reduced growth performance and, consequently, feed costs per kg of live weight gain were reduced. In Experiment 1, phytase increased (p<0.001) nitrogen-corrected AME (AMEn) from 15.39 to 15.89 MJ/kg dry matter. For nitrogen (N) retention there was an interaction (p<0.05) between diet type and phytase as the effects of phytase on N retention were more pronounced in the modified diets, with an increase from 0.512 to 0.561. These results demonstrate the positive effects of phytase on protein and energy utilisation, in addition to its established liberation of phytate-bound P and illustrate the feasibility of assigning nutrient replacement values to the feed enzyme for consideration in least-cost ration formulations. Further work is, however, required to define the most appropriate reductions in nutrient specifications in association with phytase supplementation.

• IMMUNE NETWORK
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• 제8권2호
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• pp.59-65
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• 2008

Background: Toll-like receptors (TLRs) detect microbial infection and can directly induce innate host defense responses, which are thought to play critical roles in protecting the tubotympanum from infection. However, little is known about the relationship between TLRs, which are related to innate immunity, and immunoglobulins, which are related to adaptive immunity, in recurrent otitis media with effusion (OME). We therefore investigated the expression of TLR2 and TLR4 and immunoglobulin in children with OME. Methods: The study population consisted of 72 children with OME, 31 with more than 4 episodes in 12 months or more than 3 episodes in 6 months (otitis-prone group), and 41 with fewer than 3 episodes in 12 months (non-otitis prone group). The expression in middle ear effusion of TLR2 and TLR4 mRNA, as determined by Real time- -polymerase chain reaction (RT-PCR), and the concentrations of IgG, IgA, and IgM, as determined by Enzyme-linked immunosorbent assay(ELISA), were compared between the two groups. Results: Expression of TLR2 and TLR4 mRNA was lower in the otitis prone than in the non-otitis prone group, but the difference was not statistically significant (p>0.05). Between group differences in the concentrations of IgG, IgA and IgM in effusion fluid were not significant (p>0.05), and there were no correlations between immunoglobulin concentration and the expression of TLR2 and TLR4. Conclusion: Although there was a trend toward lower expression of TLR2 and TLR4 in the otitis-prone group, the differences, and those in immunoglobulin concentration, did not differ significantly between the otitis-prone and non-prone groups.

• Restorative Dentistry and Endodontics
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• 제19권2호
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• pp.372-383
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• 1994

Saliva plays an important role in modulating the oral microbial ecology. And it is suggested to influence the initiation and progression of the dental caries. To evaluate the correlations between the salivary antimicrobial agents and the caries susceptibility, the 51 subjects were divided into 3 groups according to caries experience ; caries resistant group, medium caries susceptible group, and high caries susceptible group. Stimulated whole saliva was collected, and the salivary levels were measured for lysozyme, lactoferrin, and secretory-IgA to Streptococcus mutans. The lysozyme level was estimated using Micrococcus diffusion plate, lactoferrin level was determined with a non-competitive avidin-biotin enzyme immunoassay, and the titer of secretory IgA to Streptococcus mutans was assayed with ELISA. The results were as follows: 1. Lysozyme levels of each group showed no significant difference statistically (p>0.05). 2. The caries resistant group and the medium caries susceptible group had significantly higher levels of lactoferrin than the high caries susceptible group (p<0.05). But no clear difference was observed between the caries resistant group and the medium caries susceptible group(p>0.05). 3. The caries resistant group and the medium caries susceptible group showed relatively higher levels of the secretory IgA to Streptococcus mutans than the pigh caries susceptible group, but no significant difference was observed statistically (p>0.05).

• Journal of Microbiology and Biotechnology
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• 제19권1호
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• pp.65-71
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• 2009

Microbial oxidoreductive systems have been widely used in asymmetric syntheses of optically active alcohols. However, when reused in multi-batch reaction, the catalytic efficiency and sustainability of non-growing cells usually decreased because of continuous consumption of required cofactors during the reaction process. A novel method for NADPH regeneration in cells was proposed by using pentose metabolism in microorganisms. Addition of D-xylose, L-arabinose, or D-ribose to the reaction significantly improved the conversion efficiency of deracemization of racemic 1-phenyl-1,2-ethanediol to (S)-isomer by Candida parapsilosis cells already used once, which afforded the product with high optical purity over 97%e.e. in high yield over 85% under an increased substrate concentration of 15 g/l. Compared with reactions without xylose, xylose added to multi-batch reactions had no influence on the activity of the enzyme catalyzing the key step in deracemization, but performed a promoting effect on the recovery of the metabolic activity of the non-growing cells with its consumption in each batch. The detection of activities of xylose reductase and xylitol dehydrogenase from cell-free extract of C. parapsilosis made xylose metabolism feasible in cells, and the depression of the pentose phosphate pathway inhibitor to this reaction further indicated that xylose facilitated the NADPH-required deracemization through the pentose phosphate pathway in C. parapsilosis. moreover, by investigating the cofactor pool, the xylose addition in reaction batches giving more NADPH, compared with those without xylose, suggested that the higher catalytic efficiency and sustainability of C. parapsilosis non-growing cells had resulted from xylose metabolism recycling NADPH for the deracemization.

• 대한바이러스학회지
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• 제30권3호
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• pp.179-187
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• 2000

The etiology of rheumatic arthritis (RA) is associated with a number of genetic and environmental factors, but is not definitively elucidated. Recently, more attention has been paid to the possibility of microbial etiology in the pathogenesis of RA, because many different infectious agents have been reported to precede the onset or exacerbation of RA. Adenovirus (ADV) may be one cause of persistent or recurrent inflammatory arthritis. Varicella zoster virus (VZV) arthritis is detected frequently in RA patients treated with low dose methotrexate. The demonstration of simultaneous presence of both viral agents of specific viral nucleic acid in synovial fluids from synovitis patients would provide more direct evidence for arthritis etiological relationship, but there are no confirmed results. Therefore, we studied the ability of adenovirus and VZV to establish coinfection and persistent infection in synovial fluid from synovitis patients. The presence of viral agents in the synovial fluid demonstrated by isolation of cell culture, enzyme immunoassay and nested-PCR. The synovial fluids were also investgated for the presence of viral nucleic acid by nested-PCR using specific primer. ADV produced 220 bp and VZV produced 447 bp by each nested-PCR with specific primers. We detected 4/6 cases (66.7%) with persistent infection of ADV and 5/6 cases (83.3%) of VZV with 13 synovial fluids (between 7 to 52 day intervals) from synovitis patients by monoclonal ErA and nested-PCR. 21/28 cases (75%) with coinfection of adenovirus and VZV with synovial fluids from synovitis patients by nested-PCR. ADV and VZV coinfection and persistent infection of synovial fluids may provide a chronic antigenic stimuli to the immune system therefore provoking a continuing inflammatory response and caused the possibility of synovitis and arthritis.