• Journal of Microbiology and Biotechnology
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• 제19권9호
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• pp.888-897
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• 2009

Lipase diversity in glacier soil was assessed by culture-independent metagenomic DNA fragment screening and confirmed by cell culture experiments. A set of degenerate PCR primers specific for lipases of the hormone-sensitive lipase family was designed based on conserved motifs and used to directly PCR amplify metagenomic DNA from glacier soil. These products were used to construct a lipase fragment clone library. Among the 300 clones sequenced for the analysis, 201 clones encoding partiallipases shared 51-82% identity to known lipases in GenBank. Based on a phylogenetic analysis, five divergent clusters were established, one of which may represent a previously unidentified lipase subfamily. In the culture study, 11 lipase-producing bacteria were selectively isolated and characterized by 16S rDNA sequences. Using the above-mentioned degenerate primers, seven lipase gene fragments were cloned, but not all of them could be accounted for by the clones in the library. Two full-length lipase genes obtained by TAIL-PCR were expressed in Pichia pastoris and characterized. Both were authentic lipases with optimum temperatures of ${\le}40^{\circ}C$. Our study indicates the abundant lipase diversity in glacier soil as well as the feasibility of sequence-based screening in discovering new lipase genes from complex environmental samples.

• Reproductive and Developmental Biology
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• 제35권4호
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• pp.485-489
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• 2011

The present study was conducted to examine the reactive oxygen species (ROS) generation levels and subsequent DNA damage in the bovine cultured somatic cells. Bovine ear skin cells were classified by serum starvation, confluence and cycling cells. Cells were stained in 10 ${\mu}M$ dichlorohydrofluorescein diacetate ($H_2DCFDA$) or 10 ${\mu}M$ hydroxyphenyl fluorescein (HPF) dye to measure the $H_2O_2$ or $^{\cdot}OH$ radical levels. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each cell. $H_2O_2$ and $^{\cdot}OH$ radical levels of cultured somatic cells were high in confluence group ($7.1{\pm}0.7$ and $8.4{\pm}0.4$ pixels/cell, respectively) and significantly low in serum starvation group ($4.9{\pm}0.4$ and $7.0{\pm}0.4$ pixels/cell, respectively, p<0.05). Comet tail lengths of serum starvation ($148.3{\pm}5.7$ ${\mu}M$) and confluence ($151.1{\pm}5.0$ ${\mu}M$) groups were found to be significantly (p<0.05) increased in comparison to that of cycling group ($137.1{\pm}7.5$ ${\mu}M$). These results suggest that the culture type of donor cells can affect the ROS generation, which leads the DNA fragmentation of the cells.

• 한국산업보건학회지
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• 제23권2호
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• pp.50-56
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• 2013

Objectives: The present study investigated cytotoxicity and DNA damage in human lung cells in vitro. Methods: Neodymium oxide and lanthanum oxide were dispersed by ultrasonic treatments. The assay was performed with MRC-5 (Human male fetus lung cell). Cytotoxicity and comet assay of lanthanum oxide and neodymium oxide were measured after 24 and 48 hours incubation. Results: After 24 hours of exposure to rare earth metals, the cytotoxicities of lanthanum oxide in more than $1{\mu}M$ concentration groups were significantly increased when compared to the control group, but the cytotoxicities of neodymiun oxide in more than $100{\mu}M$ concentration groups were statistically increased. After 48 hours exposure, cytotoxicities of both materials were statistically increased in $100,000{\mu}M$ concentration groups. Olive tail moments of the lanthanum oxide treated group were significantly increased when compared to the control group. Conclusions: The cytotoxicity of lanthanum oxide was higher than that of neodymium oxide. The DNA of MRC-5 cells treated with lanthanum oxide for 48 hours were significantly damaged.

• 대한한방내과학회지
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• 제42권6호
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• pp.1269-1284
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• 2021

Objectives: Phragmitis Rhizoma is the fresh or dried rhizome of Phragmites communis Trin., which has been prescribed in traditional Korean medicine to relieve fever and vomiting and to nourish the body fluids. Recently, the protective effect of Phragmitis Rhizoma extract or its components on myelotoxicity and inflammatory responses have been reported, but no study has yet been conducted on oxidative stress. Methods: The present study investigated whether an ethanol extract of Phragmitis Rhizoma (PR) could protect against cellular damage induced by oxidative stress in Chang liver cells. Results: Pretreatment with PR significantly suppressed the hydrogen peroxide (H₂O₂)-induced reduction of Chang cell viability and generation of reactive oxygen species (ROS), thereby deferring apoptosis. PR also markedly inhibited H₂O₂-induced comet tail formation and phospho-γH2AX expression, suggesting that PR protected against oxidative stress-mediated DNA damage. PR also effectively prevented the inhibition of ATP synthesis in H₂O₂-treated Chang cells by inhibiting the loss of mitochondrial membrane potential, indicating that PR maintains energy metabolism through preservation of mitochondrial function while eliminating ROS generated by H₂O₂. Immunoblotting results indicated that PR attenuated the H₂O₂-induced downregulation of Bcl-2 and upregulation of Bax expression. Conclusions: PR protects against oxidative injury in Chang liver cells by regulating energy homeostasis via ROS generation blockade, which is at least partly mediated through inactivation of the mitochondria-mediated apoptosis pathway.

• Fisheries and Aquatic Sciences
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• 제6권4호
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• pp.180-186
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• 2003

The full-length cDNA encoding the pre-protein growth hormone (sfGH) from spotted flounder (Verasper variegatus) was amplified by the rapid amplification of cDNA ends (RACE) using degenerated oligonucleotide primers derived from conserved growth hormone sequences. It consists of 901 nucleotides in length, including the coding region of 609 nucleotides, 111 nucleotides of a 5' untranslated region, and 181 nucleotides of a 3' untranslated region. The conserved polyadenylation signal (AATAAA) lies 12 bases upstream from the poly (A) tail. The deduced amino acid sequence shows an open reading frame encoding a pre-protein of 203 amino acids and a putative signal peptide of 17 amino acids, suggesting that the mature hormone consists of 186 amino acids. The analyses of sfGH reveal some unique structural features. The repetitive sequences are located in the 5' untranslated region of sfGH cDNA and consist of tandem arrays of imperfect direct repeat monomers. Moreover, sfGH contains six Cys residues, as opposed to four or five in other GHs, and it is clearly distinguishable from olive flounder (Paralichthys olivaceus) GH, which lacks a region corresponding to residues 175-188 in alignment positions. It has important implications from an evolutionary standpoint, suggesting possible divergence among flatfishes.

• 한국환경성돌연변이발암원학회지
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• 제24권2호
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• pp.79-84
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• 2004

Three synthetic chemicals, benzoyl chloride, 2-propyn-l-ol, and 2-phenoxy ethanol were selected for genotoxicity testing, based on production quantity and available genotoxic data. In our previous report, benzoyl chloride induced chromosomal aberrations in Chinese hamster lung (CHL) fibroblast in vitro with and without metabolic activation, while 2-propyn-l-ol and 2-phenoxy ethanol induced only with metabolic activation. To compare the genotoxicity of chromosome aberration assay, the single cell gel electrophoresis (comet) assay subjected using CHL cells. As a result, statistically significant differences of tail moment values of benzoyl chloride, 2-propyn-1-ol, and 2-phenoxy ethanol were observed compared with control values on almost all concentrations with S9 or without S9 metabolic activation system. This results suggest that genotoxic results of the comet assay and the chromosome aberration assay show correlationship of genotoxicity in the CHL fibroblast. In summary, the positive result of chromosome aberration of benzoyl chloride, 2-propyn-l-ol, and 2-phenoxy ethanol was also induced DNA damages in comet assay with same cell line. Consequently, comet assay will be useful and more accurate tool to detect and to confirm the genotoxicity especially DNA damages in CHL fibroblast.

• 동아시아식생활학회지
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• 제16권4호
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• pp.399-407
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• 2006

We evaluated the effects of butterbur (Petasites japonicus Maxim) addition to the diet on lipid profiles and antioxidant biomarkers such as total glutathionine, TBARS value, carbonyl value, GPx, GR, SOD and paraoxonase activity in the plasma or liver of mice. The distribution of body fat deposition, total cholesterol (TC) contents, and atherogenic index in the plasma were significantly decreased in the butterbur group. The levels of GSH and the activity of GR and SOD were significantly higher in the liver of the butterbur group than in that of the control group. Lipid oxidation of the liver and kidney and protein oxidation of the liver and heart were decreased in the butterbur group. Additionally, the DNA damage, as determined using the comet assay (single cell gel assay) with alkaline electrophoresis and as quantified by measuring the tail length (TL), was decreased in the butterbur group. The results of the present study showed that a diet with added butterbur exerts degenerative disease-protective effects on oxidative DNA damage and lipid peroxidation.

• The Plant Pathology Journal
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• 제24권3호
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• pp.361-364
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• 2008

The complete nucleotide sequences of a Korean isolate of Potato virus X(PVX-Kr) has been determined. Full-length cDNA of PVX-Kr has been directly amplified by long template reverse transcription and polymerase chain reaction(RT-PCR) using virus specific 5'-end primer and 3'-end primer, and then constructed in a plasmid vector. Consecutive subclones of a full-length cDNA clone were constructed to identify whole genome sequence of the virus. Total nucleotide sequences of genome of PVX-Kr were 6,435 excluding one adenine at poly A tail, and genome organization was identical with that of typical PVX species. Comparison of whole genome sequence of PVX-Kr with those of European and South American isolates showed 95.4-96.8% and 77.4-77.9%, in nucleotide similarity, respectively. Sequenced PVX-Kr in this study and twelve isolates already reported could be divided into two subgroups in phylogeny based on their complete nucleotide sequences. Phylogenetic tree analysis demonstrated that PVX-Kr was clustered with European and Asian isolates(Taiwan, os, bs, Kr, S, X3, UK3, ROTH1, Tula) in the same subgroup and South American isolates(CP, CP2, CP4, HB) were clustered in the other subgroup.

• 생명과학회지
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• 제17권11호
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• pp.1497-1504
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• 2007

사람의 혈소판조절인자 (TPO)의 분비와 기능을 분석하기 위하여 사람 간 cDNA로부터 TPO cDNA를 분리하여 동물세포에서 재조합체를 생산하였다. 또한, 당쇄의 기능분석을 위하여 6개의 당쇄첨가부위를 Ala으로 치환하여 각각의 돌연변이체 재조합체도 생산하여 이들의 생리활성분석을 위하여 피하주사하여 혈소판의 증가여부를 분석하였으며, 체내 약동학검사를 위하여 꼬리 정맥에 재조합체를 주사하여 24시간까지 혈액을 채취하였다. Wild-type TPO는 효과적으로 분비하였으나, 크로닝에서 분석되어 진 116개 아미노산이 삭제된 돌연변이체는 배양상층으로 분비되지 않았다. N-linked 당쇄첨가 부위는 3번과 4번을 제외하고는 거의 비슷한 발현양상을 나타내었다. 특히 4번당쇄부위는 TPO의 분비에 중요한 역할을 하는 것으로 나타났다. 재조합체 10ng을 피하주사에 의하여 체내 혈소판이 유의적으로 증가하였으며, 5ng을 이용한 약동학 분석결과 1시간에 최대로 증가하였으며 그 이후 급격하게 감소하여 10시간에는 거의 존재하지 않았다. 따라서, 이러한 연구는 고 활성을 가지는 유전자 재조합체 TPO의 생산을 가능하게 하고, 또한 새로운 분자의 TPO를 가능하게 할 것으로 사료된다.

• 한국식품영양과학회지
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• 제33권8호
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• pp.1294-1301
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• 2004

본 연구에서는 전립선암 환자를 대상으로 이소플라본 보충제 섭취가 혈중 PSA 농도, 지질 패턴, 면역체계 및 항산화체계에 미치는 영향에 대하여 검토하고자 하였다. 전립선암환자에 게 하루 150 mg의 이소플라본을 섭취시킨 결과, 이소플라본 섭취 전에 비해 소변 내 이소플라본의 배설량이 유의적으로 증가하였다. PSA(prostate specific antigen)의 경우는 이소플라본 섭취 전보다 섭취 2달 후에 약간 증가함을 보였으나 유의적인 수준은 아니었다. 이소플라본 섭취가 전립선암 환자의 혈청 지질패턴에 미치는 영향에 대한 분석결과, 총 콜레스테롤의 경우 이소플라본 섭취 시작 전에 비해 섭취 2달 후에 유의적으로 감소되었으며, LDL 콜레스테롤의 경우 이소플라본 섭취 2달 후에 감소하는 경향을 보였으나 유의적인 차이를 보이지 않았고, HDL 콜레스테롤 수준에는 변화가 없는 것으로 나타났다. IL-6의 경우, 이소플라본 섭취 전에 비해 이소플라본 섭취 후에 수치가 감소하였고, VEGF와 TNF-$\alpha$는 이소플라본 섭취 전보다 섭취 2개월 후에 증가하였으나 유의적인 차이를 보이지는 않았다. 이소플라본 섭취전ㆍ후의 총항산화력을 TAS를 통해 측정한 결과 이소플라본 투여 전에 비 해 투여 2개월 후 수치가 증가하였으나 통계적으로 유의적인 차이는 보이지 않았다. 또한 혈액에서 분리된 임파구의 DNA 손상 정도를 측정하기 위해 comet assay를 시도한 결과 tail moment (TM)의 경우 이소플라본 섭취 2달 후에 감소하였으나 유의적인 차이는 없었으며, tail length(TL)의 경우는 유의적으로 감소하였다. 본 연구의 제한점으로는 연구 대상자의 수가 적었으며, 실험 결과를 비교할 대조군이 없었고, 연구기간이 짧다는 것을 들 수 있다. 따라서 이번 연구에서 이소플라본이 전립선암에 기대되는 효과를 보여주기엔 미비한 점이 있었다. 그러나 본 연구결과 이소플라본은 전립선암 환자의 혈중 지질 패턴과 항산화체계를 개선시키는 효과를 보였다. 따라서 이소플라본이 지질대사에 미치는 영향과 항산화작용기전에 대한 보다 심층적인 연구가 이루어져야 할 것이며, 보다 많은 전립선암 환자를 대상으로 이소플라본 섭취에 따른 장기적인 임상실험연구가 이루어져 이소플라본이 전립선암 환자의 삶의 질을 향상시킬 수 있는지에 대한 다각적인 검토가 필요하다고 본다.