• Preventive Nutrition and Food Science
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• 제9권3호
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• pp.283-288
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• 2004

Superoxide dismutase (SOD) from tomato (Lycopersicon esculentum Mill.) fruit was purified by ammonium sulphate precipitation, Sephadex G-100 and DEAE-cellulose column chromatographies. A 22 fold purification and an overall yield of 44% were achieved. The purified enzyme was a homodimer with Mr 37.1 kDa and subunit Mr 18.2 kDa as judged by SDS-PAGE. SOD showed $K_{m}$ values of 25 ${\times}$ 10$^{-6}$ M and 1.7 ${\times}$ 10$^{-6}$ M for nitroblue tetrazolium (NBT) and riboflavin as substrates, respectively. The enzyme was thermostable upto 5$0^{\circ}C$ and exhibited pH optima of 7.8. The effect of metal ions and some other compounds on enzyme activity was studied. $Co^{2+}$ and $Mg^{2+}$ were found to enhance relative enzyme activities by 27 % and 73 %, respectively, while M $n^{2+}$ inhibited the SOD activity by 64%. However, $Ca^{2+}$ and C $u^{2+}$ had no effect on enzyme activity. Other compounds like $H_2O$$_2$ and Na $N_3$ inhibited enzymatic activities by 60% and 32%, respectively, while sodium dodecyl sulphate (SDS), chloroform plus ethanol and $\beta$-mercaptoethanol had no effect on the activity of SOD. of SOD.

• 한국수정란이식학회지
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• 제19권2호
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• pp.173-183
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• 2004

본 연구는 pFF, cysteine, ${\beta}$-mercaptoethanol, 성선자극호르몬 등 여러 가지 체외성숙 촉진 물질이 첨가딘 성숙배양액에 EGF 첨가가 돼지 미성숙 난포란의 체외성숙에 효과적인지 또한 그 효가는 배양소적당 COC의 수에 영향을 받는지을 구명하고자 EGF의 첨가 유무와 배양소적당 COC수(50개 또는 15개)를 조합한 $2{\times}2$ 요인시험을 실시했다. 도축돼지의 난소에서 채취한 COCs를 각 처리별로 mNCSU-23 에서 성숙배양하고 mTBM에서 운동정자의 최종동도가 $1{\times}10^5$sperm/mL 의 농도로 체외수정한 다음 NCSU-23 에서 체외발달을 유도한 결과는 다음과 같다. 1. 대조구와 EGF 첨가구에서 C4군의 COC 비율이 각각 41%dhk 82%로써 EGF 첨가가 난구세포의 팽화 정도를 크게 향상시키는 것으로 나타났으나, 난핵포 붕괴율과 핵성숙율에서는 EGF 첨가 여부나 배양소적당 COC 수에 따른 차이가 나타나지 않았고 두 요인간 상호작용도 없었다. 2. 정자 침투율, 웅성전핵 형성율 및 다정자수정 발생율에서도 EGF 첨가 여부나 배양소적당 COC 수에 따른 차이가 유의적인 것이 아니었고, 두 요인간 상호작용 역시 유의적이지 않았다. 3. 난분할율에서는 처리간에 유의적인 차이가 나타나지 않았으나 배반포 발달율, 배반포의 ICM 세포수, TE 세포수 및 총세포수에서는 EGF 첨가구가 모두 유의적으로 높았다(p<0.01). COC수에 따른 차이나 두 요인간 상호작용에서는 유의성이 인정되지 않았다. 결과적으로 돼지 미성숙 난포란을 mNCSU-23에서 성숙배양할 때 10 ng/mL EGF 첨가는 성숙배양 단계에서 난구세포의 팽화를 더욱 심화시키고, 발달배양단계에서 배반포의 세포수와 배반포발달율을 증가시키는데 효과가 있는 것으로 사료된다.

• Reproductive and Developmental Biology
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• 제36권2호
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• pp.115-120
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• 2012

This study was conducted to examine the optimal concentration and treatment time of antioxidants for inhibition of the ROS generation in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine oocytes were activated parthenogenetically, during which oocytes were treated with various antioxidants to determine the optimal concentrations and kind of antioxidants. Determined antioxidants were applied to oocytes during in vitro maturation (IVM) and/or SCNT procedures. Finally, antioxidant-treated SCNT embryos were compared with in vitro fertilized (IVF) embryos. $H_2O_2$ levels were analyzed in embryos at 20 h of activation, fusion or insemination by staining of embryos in $10{\mu}M$ 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA) dye, followed by fluorescence microscopy. $H_2O_2$ levels of parthenogenetic embryos were significantly lower in $25{\mu}M$ ${\beta}$-mercaptoethanol (${\beta}$-ME), $50{\mu}M$ L-ascorbic acid (Vit. C), and $50{\mu}M$ L-glutathione (GSH) treatment groups than each control group ($24.0{\pm}1.5$ vs $39.0{\pm}1.1$, $29.7{\pm}1.0$ vs $37.0{\pm}1.2$, and $32.9{\pm}0.8$ vs $36.3{\pm}0.8$ pixels/embryo, p<0.05). There were no differences among above concentration of antioxidants in direct comparison ($33.6{\pm}0.9{\sim}35.2{\pm}1.1$ pixels/embryo). Thus, an antioxidant of $50{\mu}M$ Vit. C was selected for SCNT. $H_2O_2$ levels of bovine SCNT embryos were significantly lower in embryos treated with Vit. C during only SCNT procedure ($26.4{\pm}1.1$ pixels/embryo, p<0.05) than the treatment group during IVM ($29.9{\pm}1.1$ pixels/embryo) and non-treated control ($34.3{\pm}1.0$ pixels/embryo). Moreover, $H_2O_2$ level of SCNT embryos treated with Vit. C during SCNT procedure was similar to that of IVF embryos. These results suggest that the antioxidant treatment during SCNT procedures can reduce the ROS generation level of SCNT bovine embryos.

• 한국수정란이식학회지
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• 제18권3호
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• pp.243-248
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• 2003

1. 생쥐 고환으로부터 얻은 세포를 배양하여 군집을 형성하는 것을 관찰할 수 있었으며, AP, SSEA-1, -3, -4과 Integrin $\alpha$6, $\beta$1 및 Oct4의 발현을 확인하였다. 2. 생쥐 생식줄기세포를 3-5일정도 배양하게 되면, 여러 층으로 이루어진 군집을 이루게 되는데 이는 생쥐 배아줄기세포나 배아생식줄기세포의 형태와 같은 것이었다. 3. 생쥐 생식줄기세포를 체외에서 효과적으로 분리, 배양할 수 있는 조건을 확립하였다.

• 한국가축번식학회지
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• 제21권4호
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• pp.345-353
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• 1997

Antioxidants and antioxidants with somatic cell co-culture, bovine oviduct epithelial cells(BOEC) and buffalo rat liver cells(BRLC), were studied as a mean of increasing the development and intracellular glutathione(GSH) concnetrations of bovine embryos derived from in vitro matured(IVM) and in vitro fertilized(IVF) oocytes. Cell numbers and intracellular GSH concentrations of blastocysts were also counted. The developmental rate beyond morula stages in CRlaa containing taurine(2.5mM), superoxide dismutase(SOD, 600U) and catalase(250U) were 1%, 75.0%, 64.8% and 62.3%, respectively. The developmental rate in antioxidant groups was significantly higher than in control(P<0.05). The intracellular GSH concentrations of blastocysts cultured in 0, 2.5mM taurine, 600U SOD and 250U catalase were 33.8pM, 39.3pM, 42.3pM and 54.8pM, respectively. This result indicated that the developmental rates and intracellular GSH concentrations of catalase group was significantly higher than any other groups(P<0.05). The developmental capacity in CRlaa plus various antioxidants co-cultured with BOEC were 55.3%(control), 74.1%(2.5mM taurine), 66.7%(600U SOD) and 70.7%(250U catalase) and in CRlaa plus various antioxidants co-cultured with BRLC in control, 2.5mM taurine, 600U SOD and 250U catalase were 63.8%, 75.5%, 71.0% and 73.5%, respectveily, the intracellular GSH concentrations of blastocyst embryos co-cultured with BOEC and BRLC in CRlaa with 0.25mM taurine, 600U SOD and 250U catalase were 73.4pM and 64.4pM, 79.9pM and 67.5pM, 82.3pM and 71.7pM, and 83.0pM and 80.0pM, respectively. Cell numbers of blastocysts were not difference in all experimental groups. These studies indicate that andtioxidants and antioxidant with somatic cell co-culture can increase the proportion of embryo that developed into morula and blastocysts, and the intracellular GSH concentrations of blastocyst embryos.

• Journal of Microbiology and Biotechnology
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• 제20권3호
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• pp.518-524
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• 2010

A mannanase gene (man26B) was obtained from a sea bacterium, Paenibacillus sp. BME-14, through the constructed genomic library and inverse PCR. The gene of man26B had an open reading frame of 1,428 bp that encoded a peptide of 475- amino acid residues with a calculated molecular mass of 53 kDa. Man26B possessed two domains, a carbohydrate binding module (CBM) belonging to family 6 and a family 26 catalytic domain (CD) of glycosyl hydrolases, which showed the highest homology to Cel44C of P. polymyxa (60% identity). The optimum pH and temperature for enzymatic activity of Man26B were 4.5 and $60^{\circ}C$, respectively. The activity of Man26B was not affected by $Mg^{2+}$ and $Co^{2+}$, but was inhibited by $Hg^{2+},\;Ca^{2+},\;Cu^{2+},\;Mn^{2+},\;K^+,\;Na^+$, and $\beta$-mercaptoethanol, and slightly enhanced by $Pb^{2+}$ and $Zn^{2+}$. EDTA did not affect the activity of Man26B, which indicates that it does not require divalent ions to function. Man26B showed a high specific activity for LBG and konjac glucomannan, with $K_m,\;V_{max}$, and $k_{cat}$ values of 3.80 mg/ml, 91.70 ${\mu}mol$/min/mg protein, and 77.08/s, respectively, being observed when LBG was the substrate. Furthermore, deletion of the CBM6 domain increased the enzyme stability while enabling it to retain 80% and 60% of its initial activity after treatment at $80^{\circ}C$ and $90^{\circ}C$ for 30 min, respectively. This finding will be useful in industrial applications of Man26B, because of the harsh circumstances associated with such processes.

• 대한핵의학회지
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• 제26권2호
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• pp.346-354
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• 1992

서울대학교 의과대학에서 개발한 항 CEA항체에 대한 방사성옥소 및 $^{99m}Tc$의 표지법을 개발하고 이런 표지항체의 면역학적 특성을 분석하였다. 1) 8가지의 항 CEA의 단세포군 항체중 CEA-79항체와 CEA-92항체가 가장 좋은 면역학적 특성을 보였다. 표지항체의 면역반응성은 60% 정도이었고, 항원/항체 친화상수는 $1\sim2\times10^9l/m$ 이었다. 2) CEA-79항체는 chloramine-T법으로, CEA-92항체는 iodogen 법으로 방사성옥소를 표지 하는 것이 가장 좋은 표지효율 및 면역 반응성을 보였다. 3) CEA-79항체는 0.5 mCi/mg에서부터 100mCi/mg까지 비방사능을 변화시켰을 때 표지효율이나 면역 반응성에 유의한 차이는 발견할 수 없었다. 4) Glucarate를 이용한 pretargeting transchelation법으로 CEA-79항체를 $^{99m}Tc$로 표지하는 조건을 확립하였다. 표지효율은 $60\sim70%$ 이었다. 본 실험을 통하여 얻은 적절한 조건의 $^{131}I$과 $^{99m}Tc$표지방법의 확립은 앞으로 방사면역신티그라피와 방사면역치료법에 유용하게 사용될 수 있을 것으로 기대된다.

• 한국가축번식학회지
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• 제17권4호
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• pp.339-346
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• 1994

정자의 구조와 기능을 이해하기 위해서는 정자세포구성분의 분리가 필요하다. 본 실험은 동결융해된 한우정액에 다양한 물리·화학적 처리를 하여 효과적인 정자세포구성분의 분리방법을 확립하고자 실시하였다. 물리적 분리방법으로는 vortexing 처리, 26 gauge needle 또는 strained 26 gauge needle을 1ml 주사기에 부착시킨 후 반복된 pumping, 동결보존액없이 반복된 동결융해처리등을 시행하였다. 또한, 화학적인 분리를 위해 trypsin, dithiothreitol, sodium dodecylsulfate, mercaptoethanol 등을 사용하였다. 모든 처리구중에서 가장 효과적인 정자두부와 미부의 절단을 strained 26 gauge needle이 부착된 주사기를 사용한 반복된 pumping에 의해 얻어졌다. 이러한 처리에 의해 95∼100%의 높은 정자구성분의 분리가 이루어졌다. 분리된 정자구성분의 두부표면의 보존여부를 알아보기 위해 250g/ml FITC-UEA 1 염색을 실시하였지만 특별한 두부표면변화는 관찰되지 않았다. 다른 물리적 처리방법들도 높은 정자구성분의 분리결과를 보여주었지만, strained 26 gauge needle를 사용한 방법에 비해서는 분리효율, 시간등 여러면에서 비효율적이었다. 화학적 처리에 의한 정자구성분의 분리결과는 물리적 처리에 비해 효과적이지 못했다. 분리된 정자두부와 미부를 각각 회수하기 위해 sucrose 용액을 2M, 1M, 0.5M, 0.25M 순으로 시험관에 넣은 후 1,000rpm에서 15분간 원심분리한 결과, 1M과 2M의 경계부분에 형성된 정자두부층을 얻을 수 있었다. 정자구성분의 효과적이고 간편한 분리방법이 본 실험에 의해 확립되어졌으며, 위의 방법에 의해 분리, 회수된 정자구성분은 생화학적 연구, 난자활성화기작의 이해등 다양한 응용연구의 기초자료로서 이용될 수 있을 것이다.

• Restorative Dentistry and Endodontics
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• 제22권1호
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• pp.76-92
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• 1997

Porpilyromonas endodontalis is specifically involved in endodontic infections. The bacterium can be isolated almost exclusively only from infected rool canals. P. gingivalis also has been implicated in endodontic infection. Pathogemcity of P. gingival is is attributed to a variety of virulence factors, especially proteases, produced by the bacterium. Importance of P. endodontalis in endodontic infection has been revealed. However, the pathogenic property of P. endodontalis has not been extensively studied. The present study was undertaken to characterize the proteolytic activity of P. endodontalis and compare the activity with that of P. gingivalis which has the most potent and diverse proteases among oral bacteria. For this purpose, culture supematants(SUP) and cell extracts(CE) were obtained from these two bacteria and were subjected to zymography using 15% polyacrylamide gel copolymerized with gelatin, type I, IV collagens or albumin. Hydrolysis of the collagens was further investigated by the cleavage assay using native type I and IV collagens in solution-phase. The results were as follows: 1. P. endodontalis apparently has a proteolytic activity that is comparable with that of P. gingivalis. 2. SUP and CE obtained from P. endodontalis and P. gingival is showed the strongest activity for gelatin, followed by type I and IV collagens, and albumin. 3. In the zymography, no noticeable difference in proteolytic activity for gelatin and albumin between the SUP and CE was observed, but in the cleavage assay using native collagens, the SUP showed a stronger collagenolytic activity than the CE. 4. The gelatinolytic activity of both the SUP and CE from these two bacteria was diminished in the presence of $CaCl_2$ or reducing agents such as ${\beta}$-mercaptoethanol and dithiothreitol(DTT). 5. Type I(calf skin and human placenta) collagenolytic activity of P. endodontalis and P. gingivalis was reduced by DTT but not affected by $CaCl_2$. The inhibitory effect of DTT, however, was reduced to some extent by $CaCl_2$. 6. Type IV collagenolytic activity of these two bacteria was not affected by $CaCl_2$ but increased to some extent in association with the reducing agents. 7. Hydrolysis of albumin by P. endodontalis and P. gingivalis was demonstrated only in the presence of the reducing agents. The overall results indicate that with respect to proteolytic activity, P. endodontalis appears to be as potent as P. gingivalis, or maybe more, and its proteolytic characteristic is similar to that of P. gingivalis. This suggests that P. endodontalis has so potent proteolytic activity that can participate by itself in endodontic infections and apical periodontitis, causing tissue destruction.

• Applied Biological Chemistry
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• 제20권1호
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• pp.26-32
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• 1977

대두(大豆)(Glycine max cultivar Gwang-gyo)의 각 성숙시기(成熟時期)에 나타나는 7S globulin을 분리하여 Davis 방법(方法)에 의한 전기영동과 PAWU용매에 의해서 유리(遊離)되는 그들의 subunit를 전기영동한 결과 7S globulin중의 복합단백질간(複合蛋白質間)에는 그 구성(構成) subunit에 유사성(類似性)이 있음을 시사하였다. 7S globulin의 복합단백질(複合蛋白質)을 DEAE-Sephadex A-50으로 크로마토그라피하여 분리하였다. 이때 pH 7.6의 인산완충액(燐酸緩衝液)에서 NaCl의 농도구배(濃度句配)가 0.28M부터 0.40M 사이에서 두 개의 분획(分劃)으로 분리되었다. 이들 명(名) 단백질(蛋白質)의 subunit를 5M urea와 1% SDS로 유리(遊離)시켜 7.5% acrylamide-PAWU gel과 5.6% acrylamide-SDS gel에서 전기영동하였다. 그 결과 subunit의 하전량(荷電量)에 의해서 분리되는 PAWU gel전기영동에서 7S globulin이 5개의 주 분리대로 분리되고 그중 2개의 분리대가 7S-A globulin과 7S-B globulin에 공유(共有)되어 있었다. 또 subunit의 분자량(分子量)에 따라서 분리되는 SDS gel 전기영동에서는 7S globulin이 7개의 주 분리대를 나타내는데 그 중에서 3개의 분리대가 7S-A와 7S-B 분획에 공유(共有)되어 있었다. 따라서 7S globulin의 복합단백질간(複合蛋白質間)에는 구성(構成) subunit간(間)에 유사성(類似性)이 있는 것으로 나타났다.