인간 Keratinocyte HaCaT 세포에서 에토포사이드 또는 과산화수소에 의해 유도되는 아폽토시스에 미치는 플라보노이드들의 영향

조쌍구,안재연,강용진,최태원,이응룡 대한암예방학회 2006 Journal of cancer prevention Vol.11 No.1

Apoptosis is a fundamental cellular activity which allows for the maintenance of physiological balance and a protective mechanism against carcinogenesis. Recently, Flavonoids, a broadly distributed class of plant pigments, were known to regulate apoptosis and considerable scientific and therapeutic interest has focused on the structure and functions of these flavonoids in cancer chemotherapy. Despite of the many studies, the pro-oxidant/anti-oxidant properties of flavonoids remain debatable, and the detailed molecular mechanisms of their effects remain largely unknown. The objective, then, of the present work was to assess the apoptosis-modulating effects of several flavonoids in human keratinocyte HaCaT cells. In this study, we treated several flavonoids to human HaCaT keratinocytes and found that 3,4’-dihydroxy flavone and eriodictyol slightly increse cell viability, although other flavonoids including keamferol, quercetin, taxifolin, apigenin, naringenin and isohamnetin showed cell growth inhibition. 3,4’-dihydroxy flavone showed anti-apoptotic effect on etoposide or H2O2-induced cell death. Treatment of cells with 3,4’- dihydroxy flavone has decreased the nuclear fragmentation, PARP or pro-caspase 3 cleavage induced by etoposide or hydrogen peroxide in HaCaT keratinocytes. Taken together, our data strongly suggest that phytochemicals such as flavonoids and etoposide differentially regulate apoptosis in human HaCaT keratinocytes. (Cancer Prev Res 11, 58-64, 2006)

Bacillus stearothermophilus로부터 Endo-xylanase 유전자의 클로닝 및 Escherichia coli에서의 발현

조쌍구,박성수,박영인,최용진 한국산업미생물학회 1992 한국미생물·생명공학회지 Vol.20 No.3

내알카리성 및 내열성 xylanase를 생산하는 토양 분리균인 Bacillus stearothermophilus의 chromosomal DNA와 pBR322 plasmid DNA의 HindⅢ 절단 DNA 단편을 ligation시켜 E. coli HB101을 형질전환, 약 5천개의 형질전환체를 얻었으며 이들 중에서 세개의 xylanase 양성 형질전환체를 분리하였다. 상기 세 xylanase 양성 형질전환체로부터 분리한 제조합 plasmid(pMG11, pMG12 및 PMG13)는 다같이 xylanase활성과 관계되는 B. stearothermophilus 유래의 동일 4kb 외래 DNA을 가지고 있었으나, pMG13은 외래 DNA의 삽입 방향만이 다름을 확인하였다. B. stearothermophilus 균주는 최소한 세개 이상의 xylanase 활성단백질을 생산하는 것으로 관찰되었으며, xylanase 양성 형질전환체는 그 중 분자량이 가장 큰 효소단백질을 생산하는 것으로 판단되었다. 한편, 형질전환체 xylanase는 xylotetraose 이상의 xylo-oligosaccharide와 xylan 기질에 작용하여 최종 산물로서 xylobiose와 xylotriose을 생산하는 endo-acting 효소로서, trans-xylosidase 활성도 가지고 있는 것으로 추측되었다. Genomic DNA of Bacillus stearothermophilus, which expressed alkalophilic and thermophilic xylanases, was partially digested with HindⅢ, cloned into pBR322, and subsequently transferred into the Escherichia coli HB101 cells. Three among 5,000 transformants screened formed clear zones around their colonies. From the functional clones, three recombinant plasmids(pMG11, pMG12 and pMG13) had been isolated, and they were identified to carry the same 4 kb HindⅢ fragment originated from B. stearothermophilus which was responsible for the xylanase activity. pMG13, however, had the foreign DNA of opposite orientation compared to the other two recombinant plasmids. This recombinant plasmid gave much lower xylanase activity. B. stearothermophilus was observed to produce at least three xylanase activities as evidenced by the PAGE-xylan zymogram. The xylanase from E. coli HB101/pMG12 was judged to correspond to the largest among the three B. stearothermophilus xylanases observed in the zymogrom. The enzyme hydrolyzed xylooligosaccharides larger than xylotriose and degraded xylan to produce xylobiose and xylotriose as major products. The xylanase was considered to have trans-xylosidase activity, too.

다양한 세포 기능 조절 기작으로서의 단백질 인산화

조쌍구,이응룡,김장용,안재연 대한암예방학회 2006 Journal of cancer prevention Vol.11 No.1

Lots of oncogenics factors including signaling molecules, reactive oxygen species, and receptor proteins are closely involved in protein phosphorylation. Protein phosphorylation is probably one of the most studied post-translational modification mechanism which is very important for regulating the activities of important regulatory proteins in cellular signaling pathways. Here, we shortly presented recent advances in the protein phosphorylation research. Despite of the many studies, more extensive and specific research tools are needed for more comprehensive understanding of the exact molecular targets and functions of the cellular kinases. Recently, several proteomics tools are developed to analyze the phosphoproteomes or kinomes and this highthroughput study on the protein phosphorylation would be very helpful for understanding the mechanisms of many cellular functions such as cell cycle, aging, cancer or neurodegeneration. For the proteomics study, more works are needed to be done with phosphopeptides, but phosphopeptides are difficult to analyse due to the poor ionising capacity under standard MALDI conditions. Several methods have been developed to deal with the low sensitivity and specificity of the phosphopeptide analysis. The optimisation of the approach is described for a standard model peptide and protein. (Cancer Prev Res 11, 1-8, 2006)

유전자 조작 , 균주분리 재조합 균주 Escherichia coli 가 생산하는 Bacillus stearothermophilus NO.236 β-Xylosidase B의 정제 및 특성

장욱진,조쌍구,최용진 ( Uck Jin Chang,Ssang Goo Cho,Yong Jin Choi ) 한국미생물생명공학회 1998 한국미생물·생명공학회지 Vol.26 No.4

재조합 균주 Escherichia coli가 생산하는 Bacillus stearothermophilus No.236 $\beta$-Xylosidase B의 정제 및 특성

장욱진,조쌍구,최용진 한국미생물 · 생명공학회 1998 한국미생물·생명공학회지 Vol.26 No.4

Bacillus stearothermophilus No.236 xylB 유전자가 삽입된 재조합 플라스미드 pKMG12를 가지고 있는 E. coli HB101 균주를 이용하여 B. stearothermophilus $\beta$-xylosidase B을 생산, 정제하고 효소의 일반특성을 조사하였다. Ammonuim sulfate 분획, DEAE-Sepharose CL-6B 이온 교환 크로마토그래피, Sephacryl S-200 및 Superdex 200HR 젤 크로마토그래피의 과정을 거쳐 정제하였으며 정제된 효소는 SDS-PAGE 및 zymogram 실험을 통해 $\beta$-xylosidase B의 단백질임을 확인하였다. 정제 $\beta$-xylosidase B는 반응액의 수소이온 농도와 온도에 매우 민감하며 최적 활성 pH 및 온도는 각각 pH 6.5와 $50^{\circ}C$로 결정되었다. $\beta$-Xylosidase 활성은 1 mM $Mn^{2+}$ 첨가에 의해 약 35% 활성화됨을 보였으나 $Ag^{+}$, $Cu^{2+}$ 및 $Hg^{2+}$ 등의 중금속이온의 존재하에서는 거의 완전한 저해를 나타내었다. 또한 본 효소는 비록 높지는 않으나 $\alpha$-arabinofuranosidase 활성도 가지고 있어 B. stearothermophilus No 236의 $\beta$-xylosidase A 효소 보다 최소한 arabinoxylan의 분해에 있어서 더 우수한 효소로 판단되며 o-nitrophenyl-$\beta$-D-xylopyranoside 기질에 대한 $K_{m}$ 값과 $V_{max}$ 값은 각각 6.43 mM과 $1.45\mu$mole/min 로 계산되었다. 한편, $\beta$-xylosidase B 분자량은 gel 여과법으로는 약 160 kDa, 그리고 SDS-PAGE에 의해서는 약 54 kDa로 측정되어 본 효소는 trimer의 구조를 가지고 있음을 알 수 있었다. $\beta$-Xylosidase B was produced by Escherichia coli HB101/pKMG12 carrying the xylB gene of Bacillus stearothermophilus No.236 on its recombinant plasmid. The $\beta$-xylosidase B produced was purified by ammonium sulfate fractionation, DEAE-Sepharose CL-6B, Sephacryl S-200 and Superdex 200 HR gel filtration. The purified enzyme showed the highest activity at pH 6.5 and 5$0^{\circ}C$. But, the enzyme was observed to be very sensitive to the pH and temperature of the reaction mixture. The enzyme was activated about 35% of its original activity in the presence of 1 mM of $Mn^{2+}$ but it was completely inhibited by $Ag^{+}$, $Cu^{2+}$and $Hg^{2+}$ions. In contrast with the $\beta$-xylosidase A, the B enzyme was found to have $\alpha$-arabinofuranosidase activity though the activity was fairly low compared with the $\alpha$-arabinofuranosidase produced from the arfI gene of the same Bacillus stearothermophilus. Therefore, $\beta$-xylosidase B is considered to be more suitable than $\beta$-xylosidase A at least for the biodegradation of arabinoxylan. The $K_{m}$ and V$_{max}$ values of the $\beta$-xylosidase B for o-nitrophenyl-$\alpha$-D-xylopyranoside were 6.43 mM and 1.45 $\mu$mole/min, respectively. Molecular mass of the enzyme was determind to be about 54 kDa by SDS-PAGE and 160 kDa by Superdex 200HR gel filtration, indicating that the functional $\beta$-xylosidase B was composed of three identical subunits.s.

재조합 균주 Escherichia coli가 생산하는 Bacillus stearothermophilus α-L-Arabinofuranosidase의 정제 및 특성

엄수정,조쌍구,최용진 한국산업미생물학회 1995 한국미생물·생명공학회지 Vol.23 No.4

Bacillus stearothermophilus의 α-arabinofuranosidase 생산 유전자 (arfI)가 클로닝된 재조합 플라스미드(pKMG11)를 가지고 있는 E. coli HB101/pKMG11 균주에 의한 α-arabinofuranosidase 생산 최적 배양 조건을 조사해 본 결과, 탄소원으로 0.5% arabinose를 첨가한 LB 배지에서 약 20시간 배양했을 때 가장 높은 생산량을 보였다. 또한 상기 배양 조건에서 다량의 효소를 생산하고 생산 α-arabinofuranosidase를 ammonium sulfate 분획, DEAE-Sepharose CL-6B ion exchange column chromatography 및 Sepharose 6B-100 gel 여과 등의 과정을 거쳐 단일 단백질로 정제하였다. 정제 효소는 pH 6.5와 55℃에서 가장 높은 효소 활성을 보였고, 자연기질로는 arabinoxylan, 합성기질은 pNPAf에만 작용하는 매우 높은 기질특이성을 보이면서 pNPAf에 대한 K_m 값은 2.99 mM, V_max 값은 0.43 μ㏖e/min(319.74 μ㏖e/min/㎎)로 측정되었다. 또한 본 효소의 pI 값은 4.5 정도였으며 분자량은 SDS-polyacrylamide gel 영동법으로는 약 108 kDa, gel 여과법에 의해서는 약 289 kDa 정도로 측정됨으로써, arfI 생산 α-arabinofuranosidase는 trimer 인 것으로 확인되었으며 N-말단 아미노산 서열은 X-Ser-Thr-Ala-Pro-Arg(?)-Ala-Thr-Met-Val-Ile-Asp-X-Ala-Phe으로 결정되었다. α-Arabinofuranosidase was produced by E. coli HB101 haboring the recombinant plasmid pKMG11 which contained the arfI gene of Bacillus stearothermophilus. The maximum production of the enzyme was observe when E. coli HB101 cells were grown at 37℃ for 20 hours in the medium containing 0.5% arabinose, 1.0% tryptone, 0.5% yeast extract, and 1% NaCl. The α-arabinofuranosidase produced was purified to homogeneity using a combination of 20∼50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion exchange column chromatography and Sepharose 61B-100 gel filtration. The purified enzyme was most active at 55℃ and pH 6.5. The K_m and V_max values of the enzyme on ρ-nitrophenyl-α-arabinofuranoside was determined to be 2.99 mM and 0.43 μ㏖e/min (319.74 μ㏖e/min/㎎), respectively. The pI value was 4.5. The molecular weight of the native protein was estimated to be 289 kDa. The SDS-polyacrylamide gel electrophoresis analysis suggested that the functional protein was a trimer of the 108 kDa identical subunits. The N-terminal amino acid sequence of the α-arabinofuranosidase was identified as X-Ser-Thr-Ala-Pro-Arg(?)-Ala-Thr-Met-Val-Ile-Asp-X-Ala-Phe.

Xylan 분해균주인 Bacillus stearothermophilus의 오탄당 이용

이효선,조쌍구,최용진 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.4

강력한 xylan 분해 토양 분리균인 Bacillus stearothermophilus의 xylan 분해 주 산물인 xylose와 arabinose등의 pentose 이용과 pentose이용에 미치는 glucose와 기타 maltose 및 cellobiose 등의 효과를 분석하였다. 본 균주는 유일 탄소원으로 glucose를 가장 효율적으로 이용하였으며 다음으로 xylose와 ribose와 같은 pentose 그리고 maltose 등의 이당류도 잘 이용하였으나 glycerol은 전혀 이용하지 못하였다. 또한 glucose와 pentose 또는 xylooligomer와의 혼합 탄소원을 첨가했을 때도 B. stearothermophilus는 glucose를 우선적으로 이용하는 순서적 이용(sequential utilization) 현상과 더불어 전형적인 diauxic growth 양상을 나타내었다. 이에 비해 maltose와 pentose 혼합기질의 경우는 동시이용(co-utilization) 현상을 보였고 cellobios와 pentose 혼합물은 순서적 이용, 그리고 xylose와 arabinose의 pentose 혼합 탄소원인 경우는 두 탄소원을 동시에 이용하였다. 이와 같은 B. stearothermophilus의 탄소원 이용 특성은 glucose 대사관련 주요 효소인 hexokinase의 구성적 생산(constitutive production)과 이에 반한 ^D-xylose isomerase, ^D-xylulokinase 및 ^L-arabinose isomerase 등의 pentose 대사 주 관련 효소의 유도 생산(induced production)과 관계되는 효소 생산 조절 기작 내지는 inducer exclusion 현상을 비롯한 catabolite regulatory mechanism에 기인하는 것으로 설명할 수 있었다. Bacillus stearothermophilus, a potent xylanolytic bacterium isolated from soil, was tested for the strain's strategies of pentose utilization and the evidence of substreate preferences. The strain metabolized glucose, xylose, ribose, maltose, cellobiose, sucrose, arabinose and xylitol. The efficacy of the sugars as a carbon and energy source in this strain was of the order named above. The organism, however, could not grow on glycerol as a sole growth substrate. During cultivation on a mixture of glucose and xylose or arabinose, the major hydrolytic products of xylan, B. stearothermophilus displayed classical diauxic growth in which glucose was utilized during the first phase. On the other hand, the pentose utilization was prevented immediately upon addition of glucose. Cellobiose was preferred over xylose or arabinose. In contrast, maltose and pentose were co-utilized, and also no preference on between xylose and arabinose. Enzymatic studies indicated that B. stearothermophilus possessed constitutive hexokinase, a key enzyme of the glucose metabolic system. While, the production of ^D-xylose isomerase, ^D-xylulokinase and ^D-arabinose isomerase essential for pentose phosphate pathway were induced by xylose, xylan, and xylitol but repressed by glucose. Taken together, the results suggested that the sequential utilization of B. stearothermophilus would be mediated by catabolite regulatory mechanisms such as catabolite inhibition or inducer exclusion.

Bacillus stearothermophilus Acetyl Xylan Esterase 유전자의 크로닝과 Escherichia coli에서의 발현

김인숙,조쌍구,최용진 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.6

토양에서 분리한 강력한 xylan 분해 균주인 Bacillus stearothermophilus의 HindIII 절단 genomic DNA 단편을 pBR322에 크로닝하여 약 4000개의 E. coli HB101 형질전환체를 얻었으며 이 중 한 개의 형질전환체가 tributyrin 분해능을 나타내었다. 본 연구에서는 상기 형질전환체를 선별, tributyrin 분해 활성 관련 유전자를 조사 분석하였던 바 본 형질전환체가 tributyrin 분해능과 관련된 약 5.1 kb의 외래 DNA가 삽입된 재조합 plasmid를 가지고 있음을 확인하고 이 plasmid를 pKMG5로 명명하였다. 다음 5.1 kb 삽입 DNA 상의 restriction site를 결정하고 subcloning을 실시하여 높은 tributyrin 분해 활성을 나타내는 새로운 재조합 균주로부터 3.5 kb의 외래 DNA를 가지는 pKMG6를 분리하였다. 또한 pKMG6에 삽입된 tributyrin 분해 활성 관련 상기 3.5 kb 외래 DNA는 Southern blotting 실험 결과, B. stearothermophilus chromosome 유래의 DNA 단편임을 확인할 수 있었으며 동시에 삽입 DNA 단편상의 tributyrin 분해 활성 관련 유전자의 발현 산물이 일종의 acetyl xylan esterase로 분류할 수 있는 xylan 분해계 효소임을 알았다. Bacillus stearothermophilus was shown to express multiple xylanolytic enzymes including acetyl xylan esterase. Genomic DNA of the strain partially digested with HindIII was ligated into theh HindIII site of pBR322, and expressed in E. coli HB010 cells in order to clone the gene for acetyl xylan esterase. One transformant among 4000 screened formed a clear zone around its colony on the LB agar supplemented with 1.0% tributyrin. The functional clone harbored the recombinant plasmid pKMG5 with an insert of 5.1 kb. Subcloning yielded the recombinant molecule pKMG6 with 3.5 kb HindIII fragment derived from the B. stearothermophilus chromosomal DNA as determined by restriction enzyme analysis and Southern hybridization. The tributyrin degrading activity produced by the cloned gene also revealed acetyl residue cleavage activity on oat spelt acetyl xylan. The results of substrate specificity confirmed that the esterase from E. coli HB101/pKMG6 was an acetyl xylan esterase.

The effects of human milk proteins on the proliferation of normal, cancer and cancer stem like cells

강남미,조쌍구,아브달아메드,이주현,배성필,한원호,이정상 한국분석과학회 2018 분석과학 Vol.31 No.6

Human breast milk (HBM) provides neonates with indispensable nutrition. The present study evaluated the anti-cancer activity of diluted and pasteurized early HBM (< 6 weeks’ lactation) on human breast cancer cell lines. The cell lines MCF7 and MDA-MB231 were exposed to 1% HBM from the 1st, 3rd, and 6th weeks of lactation and exhibited reduced proliferation rates. As controls, breast cell lines (293T and MCF-10A), breast cancer cell lines (MCF-7 and MDA-MB-231), and CD133hiCXCR4hiALDH1hi patient-derived human cancer stem-like cells (KUCSLCs) were treated with prominent milk proteins β-casein, κ-casein, and lactoferrin at varying doses (10, 50, and 100 μg) for 24 or 48 hrs. The impact of these proteins on cell proliferation was investigated. Breast cancer cell lines treated with κ-casein and lactoferrin exhibited significantly reduced viability, in both a dose- and time-dependent manner. Interestingly, κ-casein selectively impacted only cancer (but not normal breast) cell lines, particularly the more malignant cell line. However, β-casein-exposed human breast cancer cell lines exhibited a significantly higher proliferation rate. Thus, κ-casein and lactoferrin appear to exert selective anti-cancer activities. Further studies are warranted to determine the mechanisms underlying κ-casein- and lactoferrin-mediated cancer cell-selective cytotoxic effects.

재조합 균주 Escherichia coli가 생산하는 Bacillus stearothermophilus $\alpha$-L-Arabinofuranosidase의 정제 및 특성

엄수정,조쌍구,최용진 한국미생물 · 생명공학회 1995 한국미생물·생명공학회지 Vol.23 No.4

$\alpha $-Arabinofuranosidase was produced by E. coli HB101 haboring the recombinant plasmid pKMG11 which contained the arfI gene of Bacillus stearothermophilus. The maximum production of the enzyme was observed when E. coli HB101 cells were grown at 37$\circ$C for 20 hours in the medium containing 0.5% arabinose, 1.0% tryptone, 0.5% yeast extract, and 1% NaCl. The $\ALPHA $-arabinofuranosidase produced was purified to homogeneity using a combination of 20-50% ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion exchange column chromatography and Sepharose 6B-100 gel filtration. The purified enzyme was most active at 55$\circ$C and pH 6.5. The K$_{m}$ and V$_{max}$ values of the enzyme on $\rho $-nitrophenyl-$\alpha $-arabinofuranoside was determined to be 2.99 mM and 0.43 $\mu $mole/min (319.74 $\mu $mole/min/mg), respectively. The pI value was 4.5. The molecular weight of the native protein was estimated to be 289 kDa. The SDS-polyacrylamide gel clectrophoresis analysis suggested that the functional protein was a trimer of the 108 kDa identical subunits. The N-terminal amino acid sequence of the a-arabinofuranosidase was identified as X-Ser-Thr-Ala-Pro-Arg( \ulcorner )-Ala-Thr-Met-Val-Ile-Asp-X-Ala-Phe.