• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.247-255
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• 2006

Yeast cell wall matrix particles are composed entirely of mannoprotein and ${\beta}-glucan$. The mannoproteins of yeast cell wall can systemically enhance the immune system. We previously purified and analyzed alkali-soluble ${\beta}-glucans$ [${\beta}$-(1,3)- and ${\beta}$-(1,6)-glucans] [10]. In the present study, a wild-type strain was first mutagenized with ultraviolet light, and the cell wall mutants were then selected by treatment with 1.0 mg/ml laminarinase (endo-${\beta}$-(1,3)-D-glucanase). Mannoproteins of Saccharomyces cerevisiae were released by laminarinase, purified by concanavalin-A affinity and ion-exchange chromatography. The results indicated that the mutants yielded 3-fold more mannoprotein than the wild-type. The mannoprotein mass of mutant K48L3 was 2.25 mg/100 mg of yeast cell dry mass. Carbohydrate analysis revealed that they contained mannose, glucose, and N-acetylglucosamine. Saccharomyces cerevisiae cell wall components, mannoproteins, are known to interact with macrophages through receptors, thereby inducing release of tumor necrosis factor alpha ($TNF-{\alpha}$) and nitric oxide. Mannoprotein tractions in the present study had a higher macrophage activity of secretion of $TNF-{\alpha}$ and nitric oxide and direct phagocytosis than positive control ($1{\mu}g$ of lipopolysaccharide). In particular, F1 and F3 fractions in mannoproteins of K48L3 enhanced and upregulated the activity of nitric oxide secretion and macrophage phagocytosis by approximately two- and four-fold, respectively.

• Parasites, Hosts and Diseases
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• 제53권4호
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• pp.385-394
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• 2015

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-${\gamma}$/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

• 생명과학회지
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• 제15권2호
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• pp.202-210
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• 2005

살모넬라는 질병을 일으키기 위해서 장내의 상피세포에 먼저 부착하여 집락을 이루게 된다. 이에 따라 살모넬라는 부착에 관여하는 세포내 몇 몇 소기관들을 합성한다. 이러한 소기관에는 type 1 fimbriae, plasmid-encoded fimbriae, long polar fimbriae 그리고 thin aggregative fimbriae 등이 있다. 본 논문에서는 type 1 fimbriae, thin aggregative fimbriae, LP fimbriae, 그리고 PE fimbriae 각각을 결손시킨 변이주와 4종류를 모두 결손시킨 변이주를 만들 수 있었다. 변이주들에 대해서 세포배양 부착성 실험을 한 결과, 각각을 결손시킨 변이주들은 몇몇 mammalian 세포주에서 야생형 살모넬라와 동일한 양상으로 부착성을 나타내었고 마우스 실험에서 야생형과 거의 마찬가지로 독성을 가지고 있었다. 반면, 4종류의 fimbriae가 모두 결손된 균주는 닭에서는 독성을 나타내었으나 마우스에서는 매우 약독화되어있음을 확인할 수 있었다. 이상의 결과로부터 오는 차이점은 닭과 비교하여 마우스에서 살모넬라의 부착을 매개하는 표면 구조에 연관성이 있음을 제시하였다.

• 한국식품영양과학회지
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• 제20권4호
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• pp.395-400
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• 1991

식육 및 야생쥐의 장으로부터 식품매개성 질병에 관여하는 Enterobacter를 분리하여 그 특징을 구명하므로 공중위생 및 식품위생학적으로 기초자료를 제시하고저 본 연구를 실시하여 다음과 같은 결론을 얻었다. Enterobacter는 우육에서 1주(E. aerogenes), 돈육에서 2주(E. aerogenes, E. intermedium), 야생쥐의 소장에서 2주(E. aerogenes, E. cloacae)가 동정되어 총 분리된 89균주에 대한 분리율은 5.62%이었다. 분리된 Enterobacter를 각종 화학기질이 첨가된 배지에서 $55^{\circ}C$, 35분간 열처리한 결과, 전반적으로 0.1M glycine, 5% mannitol 그리고 5% sorbitol이 첨가된 배지에서는 열저항성이 강하였고 1% sodium citrate, 1% casein, 10% NaCl 그리고 0.1M cystein이 첨가된 배지에서는 열저항성이 약하였다.

• 한국양식학회지
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• 제21권1호
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• pp.13-18
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• 2008

Marine birnavirus(MABV)는 중요한 어류 병원체로서 일본 양식산 방어서 처음 분리되어 이후 다양한 해산어종에서 MABV 분리가 보고되었으며 환경적인 샘플인 저질, 해양의 동물성 플랑크톤과 해수에서도 MABV 유전자가 검출되고 있다. 본 연구는 Marine birnavirus disease 에 대한 예방학적 접근의 일환으로서 자연산 어류로부터의 MABV 검출 및 지리적 분포, 보균 어종에 대한 유전학적인 조사를 목적으로 2003년과 2005년도에 동중국해역 3지점, 서해안 5지점 그리고 남해안 5지점에서 어류를 채집하였다. RT-PCR(Reverse transcriptase-Polymerase Chain Reaction)을 이용한 연구결과 자연산 해산어류 160마리 중 13종 17마리에서 MABV가 거출되어 감염율10.6%를 보였다. 조사된 13종의 어류 중 특히 농어목에서 가장 높는 검출율을 보였다. 자연산 어류에서 분리한 MABV 분리균주는 염기서열 분석에서 94.7%-100%, 아미노산 분석에서 97.2-100% 유사성을 나타내었으며 IPNV strain과는 구분되며 MABV에 속하였다.

• Journal of Microbiology
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• 제33권4호
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• pp.339-343
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• 1995

We isolated a 10 kb Bam HI fragment originated from the chromosome of a $H_2O$$^2$-resistant mutant strain of Streptomyces coelicolor, which confer $H_2O$$^2$-resistance to S. lividance upon transformation. Among various subclones ot 10kb Bam HI fragment tested for their $H_2O$$^2$-resistant phenotype in S. lividans, a subclone containing 5.2 kb Bam HI-BglII fragment was found to be responsible for $H_2O$$^2$-resistance. The plasmid containing this 5.2 kb fragment was then transformed into S. coellicolor A3(2) at early and tested for their phenotype of $H_2O$$^2$-resistance and the change in various enzymes whose activity can be stained in the gel. We found out that the 5.2 kb insert DNA conferred $H_2O$$^2$-resisstance in S. coelicolor A3(2) at early phase of cell growth. The presence of this DNA also resulted in higher level of peroxidase compared with the wild type cell containing parental vector (pIJ702) only. Esterase activity was also higher in this clone. However, alcohol dehydrogenase activity decreased compared with the wild type. These results suggest that the presence of a gene in 5.2 kb BamHI-BglII DNA fragment causes multiple changes in S. coelicolor related to its response against hydrogen peroxide. The result also implies that not only peroxidase but also esterase may function in the defencse meahsnism agianst $H_2O$$^2$-.

• Journal of Microbiology and Biotechnology
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• 제29권3호
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• pp.367-372
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• 2019

Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O-phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'-dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP, in the genome of wild-type Streptomyces tenjimariensis, the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3-O-methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP) in the ${\Delta}forP$ mutant (or ${\Delta}istP$ mutant strain) successfully restored the biosynthesis of 3',4'-dideoxy fortimicins and istamycins, thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.

• Journal of Microbiology and Biotechnology
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• 제30권12호
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• pp.1876-1884
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• 2020

Ethanol often accumulates during the process of wine fermentation, and mitophagy has critical role in ethanol output. However, the relationship between mitophagy and ethanol stress is still unclear. In this study, the expression of ATG11 and ATG32 genes exposed to ethanol stress was accessed by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). The result indicated that ethanol stress induced expression of the ATG11 and ATG32 genes. The colony sizes and the alcohol yield of atg11 and atg32 were also smaller and lower than those of wild type strain under ethanol whereas the mortality of mutants is higher. Furthermore, compared with wild type, the membrane integrity and the mitochondrial membrane potential of atg11 and atg32 exhibited greater damage following ethanol stress. In addition, a greater proportion of mutant cells were arrested at the G1/G0 cell cycle. There was more aggregation of peroxide hydrogen (H2O2) and superoxide anion (O2•-) in mutants. These changes in H2O2 and O2•- in yeasts were altered by reductants or inhibitors of scavenging enzyme by means of regulating the expression of ATG11 and ATG32 genes. Inhibitors of the mitochondrial electron transport chain (mtETC) also increased production of H2O2 and O2•- by enhancing expression of the ATG11 and ATG32 genes. Further results showed that activator or inhibitor of autophagy also activated or inhibited mitophagy by altering production of H2O2 and O2•. Therefore, ethanol stress induces mitophagy which improves yeast the tolerance to ethanol and the level of mitophagy during ethanol stress is regulated by ROS derived from mtETC.

• 한국버섯학회지
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• 제20권2호
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• pp.43-49
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• 2022

Pleurotus ostreatus is a globally cultivated mushroom crop. Cap color is a quality factor in P. ostreatus. However, cap color can spontaneously mutate, degrading the quality of the mushroom on the market. Early detection and removal of mutant strains is the best way to maintain the commercial value of the crop. To detect the cap color mutant Gonji7ho, molecular markers were developed based on insertion/deletions (InDels) derived from the comparison of mitogenomes of Gonji7ho and Gonji7hoM mushrooms. Sequencing, assembly, and comparative analysis of the two mitogenomes revealed genome sizes of 73,212 bp and 72,576 bp with 61 and 57 genes or open reading frames (ORFs) in P. ostreatus Gonji7ho and Gonji7hoM, respectively. Fourteen core protein-encoding genes, two rRNA, and 24 tRNA with some OFRs were predicted. Of the 61 genes or OFRs in the wild type, dpo, rpo, and two orf139 were missing (or remnant) in the mutant strain. Molecular markers were developed based on the sequence variations (InDels) between the two mitogenomes. Six polymorphic molecular markers could detect the mutated mitochondria by PCR. These results provide basic knowledge of the mitogenomes of wild-type and mutant P. ostreatus, and can be applied to discriminate mutated mitochondria.

• 생명과학회지
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• 제25권11호
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• pp.1290-1297
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• 2015

에탄올 생산 세균 Zymomonas mobilis에서 에탄올 생산 경로의 핵심으로 작용하는 효소인, pyruvate decarboxylase(pdc) 유전자의 불활성 실험을 통해, PDC 활성이 50% 감소된 PDC 활성 변형균주가 분리되었다. 이러한 균주들의 에탄올 탄소대사 흐름이 고부가가치 화합물인 피루브산, 숙신산 및 젖산 등으로 전환되는지를 발효 실험을 통해 평가하였다. 하지만 pdc의 발현을 중지시키기 위해 cat-삽입형-pdc와 pdc-결손형 아형 유전자를 전기천공법을 이용해 야생형 균주 ZM4의 염색체에 이식하기 위한 다수의 시도에도 불구하고, 이러한 방법을 통해 분리된 균주들은 대부분 부분적 유전자 불활성 특성을 보였으며, PDC 활성이 완전히 손실된 삭제 돌연변이 균주를 획득할 수는 없었다. PDC활성이 변형된 돌연변이 균주의 발효 실험에서, 야생형 균주와 비교 시 감소된 PDC 효소 활성의 변화로 인해 기질 흡수율과 에탄올 생산율이 감소되어 피루브산 생산이 약 2.5 g l^-1 정도로 증가함을 확인하였으나, 젖산과 숙신산의 생산에 현저한 농도 변화를 보이지 못했다. 이러한 결과는 Z. mobilis의 산화환원 에너지가 PDC 효소 활성에 의한 에탄올 생산 경로에 전적으로 의존하여 발생한다는 것을 암시하였다. 상기 결과를 토대로 pdc 유전자의 완전한 불활성 유도와 산화환원 에너지의 균형은, 젖산 생산을 위한 lactate dehydrogenase, 숙신산 생산을 위한 pyruvate dehydrogenase와 malic enzyme과 같은 효소의 활성 증가를 통해, 세포내 NAD와 NADH 농도의 산화환원 균형이 이루어져야 발생할 수 있음을 시사하였다.