• Journal of Microbiology and Biotechnology
• /
• 제23권3호
• /
• pp.304-312
• /
• 2013

The thermotolerant methylotrophic yeast Hansenula polymorpha is attracting interest as a potential strain for the production of recombinant proteins and biofuels. However, only limited numbers of genome engineering tools are currently available for H. polymorpha. In the present study, we identified the HpPOL3 gene encoding the catalytic subunit of DNA polymerase ${\delta}$ of H. polymorpha and mutated the sequence encoding conserved amino acid residues that are important for its proofreading 3'${\rightarrow}$5' exonuclease activity. The resulting $HpPOL3^*$ gene encoding the error-prone proofreading-deficient DNA polymerase ${\delta}$ was cloned under a methanol oxidase promoter to construct the mutator plasmid pHIF8, which also contains additional elements for site-specific chromosomal integration, selection, and excision. In a H. polymorpha mutator strain chromosomally integrated with pHIF8, a $URA3^-$ mutant resistant to 5-fluoroorotic acid was generated at a 50-fold higher frequency than in the wild-type strain, due to the dominant negative expression of $HpPOL3^*$. Moreover, after obtaining the desired mutant, the mutator allele was readily removed from the chromosome by homologous recombination to avoid the uncontrolled accumulation of additional mutations. Our mutator system, which depends on the accumulation of random mutations that are incorporated during DNA replication, will be useful to generate strains with mutant phenotypes, especially those related to unknown or multiple genes on the chromosome.

• International Journal of Oral Biology
• /
• 제45권1호
• /
• pp.15-24
• /
• 2020

The fruit of Chaenomeles sinensis (Thouin) Koehne (Chaenomelis Fructus) known as "Mo-Gua" in Korea has been commonly used in traditional medicine to treat inflammatory diseases, such as sore throat. However, its effect on bone metabolism has not been elucidated yet. Here, we examined the effect of Chaenomelis Fructus ethanol extract (CF-E) on receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated osteoclast differentiation and formation. CF-E considerably inhibited osteoclast differentiation and tartrate-resistant acid phosphatase-positive multinuclear cell formation from bone marrow-derived macrophages and osteoclast precursor cells in a dose-dependent manner. In addition, the formation of actin rings and resorption pits were significantly suppressed in CF-E-treated osteoclasts as compared with the findings in non-treated control cells. Consistent with these phenotypic inhibitory results, the expressions of osteoclast differentiation marker genes (Acp5, Atp6v0d2, Oscar, CtsK, and Tm7sf4) and Nfatc1, a pivotal transcription factor for osteoclastogenesis, were markedly decreased by CF-E treatment. The inhibitory effect of CF-E on RANKL-induced osteoclastogenesis was associated with the suppression of NFATc1 expression, not by regulation of mitogen-activated protein kinases and NF-κB activation but by the inactivation of phospholipase C gamma 1 and 2. These results indicate that CF-E has an inhibitory effect on osteoclast differentiation and formation, and they suggest the possibility of CF-E as a traditional therapeutic agent against bone-resorptive diseases, such as osteoporosis, rheumatoid arthritis, and periodontitis.

• 한국응용곤충학회지
• /
• 제24권4호
• /
• pp.219-222
• /
• 1986

우리나라의 주요(主要) 벼흰잎마름병 저항성(抵抗性) 품종(品種)의 저항성(抵抗性) 유전양식(遺傳樣式)과 저항성(抵抗性) 품종간(品種間)의 대립관계(對立關係)를 분석(分析)하기 위하여 연구(硏究)한 결과(結果)를 요약(要約)하면 다음과 같다. 1. JB8206균주(菌株)에 대(對)한 청청(靑靑)벼, 영풍(永豊)벼, 남풍(南豊)벼, 삼강(三剛)벼, 한강(漢江)찰벼의 흰 잎마름병 저항성(抵抗性)은 1개(個)의 우성유전자(優性遺傳子)에 의(依)해 지배(支配)되었다. 2. JB8206, KN8298균주(菌株)에 대(對)한 백양(白羊)벼와 풍산(豊産)벼의 저항성(抵抗性)은 1개(個)의 우성유전자(優性遺傳子)에 의(依)해 지배(支配) 되었다. 3. JB8206균주(菌株)에 대(對)한 풍산(豊産)벼와 청청(靑靑)벼, 밀양(密陽) 30호(號), 백운(白雲)찰벼의 저항성(抵抗性) 및 수원(水原)312호(號)와 백양(白羊)벼, 백운(白雲)찰벼의 저항성(抵抗性)은 대립관계(對立關係)이었으며 동일유전자(同一遺傳子)로 추정(推定)되었다.

• Archives of Pharmacal Research
• /
• 제29권8호
• /
• pp.691-698
• /
• 2006

Although glucocorticoids are known to affect osteoclast differentiation and function, there have been conflicting reports about the effect of glucocorticoids on osteoclast formation, leading to the assumption that microenvironment and cell type influence their action. We explored the effect of the synthetic glucocorticoid analog dexamethasone on the formation of osteoclasts. Dexamethasone inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts without affecting the formation of TRAP-positive mononuclear cells in a coculture of mouse osteoblasts and bone marrow cells. Dexamethasone did not inhibit mRNA expression levels of the receptor activator of nuclear factor-kB ligand and osteoprotegerin, the essential regulators of osteoclastogenesis. Dexamethasone down-regulated the expression of ${\beta}_3$ integrin mRNA and protein but did not alter expression of other osteoclast differentiation marker genes. Both dexamethasone and echistatin, a ${\beta}_3$ integrin function blocker, inhibited TRAP-positive multinucleated osteoclast formation but not TRAP-positive mononuclear cell formation. These results suggest that dexamethasone inhibits the formation of multinucleated osteoclasts, at least in part, through the down-regulation of ${\beta}_3$ integrin, which plays an important role in the formation of multinucleated osteoclasts.

• 한국응용곤충학회지
• /
• 제24권2호
• /
• pp.79-83
• /
• 1985

벼멸구에 대(對)한 벼품종간(品種間)의 피해(被害)와 수량(收量)을 조사(調査)하였다. 1983 년(年) 전남(全南) 해남(海南)에서 29개(個) 벼품종(品種)을 살충제무산포구(殺蟲劑無散布區), 살충(殺蟲) 및 살충제무산포구(殺蟲劑無散布區), 살균제무산포(殺菌劑無散布) 두어 이앙(移秧)하고 충밀도(蟲密度), 벼고사시기(枯死時期), 수량(收量)을 조사(調査)하였다. 삼강(三剛)벼, 가야(伽倻)벼, 한강(漢江)찰벼, 청청(靑靑)벼는 극히 낮은 밀도(密度)와 피해(被害)가 없었고 밀양(密陽) 30호(號)와 원풍(圓豊)벼는 중간정도(中間程度)의 밀도(密度)와 피해(被害)를 보였고 그 이외(以外) 품종(品種)은 높의 밀도(密度)와 고사현상(枯死現狀)을 나타냈다. 살충제무산포(殺蟲劑無散布)에 의한 수량(收量) 감수효과(減收效果)가 적었던 품종(品種)은 가야(伽倻)(12%), 삼강(三剛)벼(29%), 한강(漢江)찰벼(35%)였고 감수효과(減收效果)가 컷던 품종(品種)은 복광(福光)벼(260%), 팔금(八錦)(223%), 송전(松前)벼(200%), 관악(冠岳)벼(200%)였다.

• Journal of Ginseng Research
• /
• 제44권2호
• /
• pp.332-340
• /
• 2020

Background: The valuable medicinal plant Panax ginseng has high pharmaceutical efficacy because it produces ginsenosides. However, its yields decline because of a root-rot disease caused by Ilyonectria mors-panacis. Because species within Ilyonectria showed variable aggressiveness by altering ginsenoside concentrations in inoculated plants, we investigated how such infections might regulate the biosynthesis of ginsenosides and their related signaling molecules. Methods: Two-year-old ginseng seedlings were treated with I. mors-panacis and I. robusta. Roots from infected and pathogen-free plants were harvested at 4 and 16 days after inoculation. We then examined levels or/and expression of genes of ginsenosides, salicylic acid (SA), jasmonic acid (JA), and reactive oxygen species (ROS). We also checked the susceptibility of those pathogens to ROS. Results: Ginsenoside biosynthesis was significantly suppressed and increased in response to infection by I. mors-panacis and I. robusta, respectively. Regulation of JA was significantly higher in I. robusta-infected roots, while levels of SA and ROS were significantly higher in I. mors-panacis-infected roots. Catalase activity was significantly higher in I. robusta-infected roots followed in order by mock roots and those infected by I. mors-panacis. Moreover, I. mors-panacis was resistant to ROS compared with I. robusta. Conclusion: Infection by the weakly aggressive I. robusta led to the upregulation of ginsenoside production and biosynthesis, probably because only a low level of ROS was induced. In contrast, the more aggressive I. mors-panacis suppressed ginsenoside biosynthesis, probably because of higher ROS levels and subsequent induction of programmed cell death pathways. Furthermore, I. mors-panacis may have increased its virulence by resisting the cytotoxicity of ROS.

• 미생물학회지
• /
• 제25권1호
• /
• pp.9-16
• /
• 1987

분리한 살리실산 자화세균 중 플라스미드블 갖는 세 균주를 션별하였다. 세 균주, KU801(pKUs, pKUS) , KUS03(pKU6 pKU9), KUS06(pKU7, pKU10)는 각각 두 개씩의 플라스미드플 가지고 있음이 전기갱동에 의해 밝혀졌고, Pseudomoηas putida로 동정되었다. 세 문주들은 모두 암피실린, 터l트라사이클린, 클로람페니콜등의 항생제에 대하여 내성을 지니며, 조사 된 방화족과 지방족 탄화수소들 중 삼리실산과 그의 중간 대사물인 카터1콜만을 이용하있다. 큰 분자량의 플라스비드(pKUS, p pKU6, pKU7)는 마이로마이신 C로 처리하였을때 큐어되며 그 빈도는 각각 0.40%, l,67%, 0.7S% 이었다. 큐어된 균주는 상리실산을 분해하지 못하였으나, 여전히 야생균주와 동일한 항생제 내성을 가지고 있었다. 살리실산 분해에 관여하는 유전자가 그들 플라스미드에 있는 것으로 판명되었다. pKU5와 pKU6의 분자량은 103, SMd, pKU7의 분자량은 101Md으로 측정되었다. SAL 플라스미드인 pKU5, pKU6, pKU7은 접합에 의해 P.putida와 P.aeruginosa로는 전달되었으나, E. coli에서는 발현되지 아니하였다.

• 미생물학회지
• /
• 제26권4호
• /
• pp.305-311
• /
• 1988

Slow growing R. japonicum R-l68 균주로부터 spectinomycin 내성 균주를 선발하고 이 Rhizobium내에 Tn-5를 도입시키기 위하여 Tn5가 함유된 E. coli WA 803/pGS9과의 conjugation을 통한 transposon mutagenesis를 실시하였다. 이때 C conjugation을 통한 Tn5 전이 빈도는 $1.0\times 10^{-5}-5.0\times 10^{-7}$ 범위 이였으며, 얻어진 transconjugant들은 spectinomycin (($100{\mu}$g/ml)과 kanamycin ($50{\mu}$g/ml)을 함유한 yeast extract-mannitol 배지에서 8-10일 배양후 colony를 형성하였다. 또한 transconjugant들은 genome상에 Tn-5를 함유하고 있음을 hybridization-을 통하여 확인하였다. 한편 nodule은 형성 하나 질소고정 활성이 없는 돌연변이주 R. japonicum RMa 75 $nod^{+}fix^{-}$ 균주를 선발하였는데 이 균주는 nodule내에 leghemoglobin이 결핍되어 있음이 확인되었다.

• Genomics & Informatics
• /
• 제11권4호
• /
• pp.245-253
• /
• 2013

A radioresistant cell line was established by fractionated ionizing radiation (IR) and assessed by a clonogenic assay, flow cytometry, and Western blot analysis, as well as zymography and a wound healing assay. Microarray was performed to profile global expression and to search for differentially expressed genes (DEGs) in response to IR. H460R cells demonstrated increased cell scattering and acidic vesicular organelles compared with parental cells. Concomitantly, H460R cells showed characteristics of increased migration and matrix metalloproteinase activity. In addition, H460R cells were resistant to IR, exhibiting reduced expression levels of ionizing responsive proteins (p-p53 and ${\gamma}$-H2AX); apoptosis-related molecules, such as cleaved poly(ADP ribose) polymerase; and endoplasmic reticulum stress-related molecules, such as glucose-regulated protein (GRP78) and C/EBP-homologous protein compared with parental cells, whereas the expression of anti-apoptotic X-linked inhibitor of apoptosis protein was increased. Among DEGs, syntrophin beta 2 (SNTB2) significantly increased in H460R cells in response to IR. Knockdown of SNTB2 by siRNA was more sensitive than the control after IR exposure in H460, H460R, and H1299 cells. Our study suggests that H460R cells have differential properties, including cell morphology, potential for metastasis, and resistance to IR, compared with parental cells. In addition, SNTB2 may play an important role in radioresistance. H460R cells could be helpful in in vitro systems for elucidating the molecular mechanisms of and discovering drugs to overcome radioresistance in lung cancer therapy.

• Journal of Microbiology and Biotechnology
• /
• 제27권9호
• /
• pp.1711-1715
• /
• 2017

In this study, we characterized the $bla_{NDM-5}$-bearing plasmid in a Klebsiella pneumoniae isolate that had lost the plasmid during serial passage. We determined the complete sequences of the plasmid pCC1410-2, which was extracted from a K. pneumoniae ST709 isolate collected at a Korean hospital from which two NDM-5-producing K. pneumoniae isolates were subsequently isolated. As a result, the pCC1410-2 plasmid had a backbone structure that was similar to those of two plasmids previously reported from the same hospital, but lacked some antibiotic resistance genes ($bla_{TEM-1}$, rmtB, mphR(A), mrx(A), and mph(A)). A 9-bp repeating unit encoding three amino acids (Gln-Gln-Pro) was inserted in TraD in pCC1410-2. Thus, the pCC1410-2 plasmid might be transferred from the previously identified carbapenem-resistant K. pneumoniae, but some delections and inversions might have occurred during the process. We compared the transfer frequency and stability of the plasmids. The relative frequency of conjugative transfer and stability in the host were significantly lower in pCC1410-2 than in previously reported $bla_{NDM-5}$-bearing plasmids in Korea. A low transfer frequency and instability in the host may cause underestimation of carbapenemase-producing Enterobacteriaceae in the clinical setting and in surveillance studies.