• 대한수의학회지
• /
• 제39권1호
• /
• pp.220-225
• /
• 1999

The aim of these experiments was to investigate the effects of bovine oviduct epithelial cells (OEC) derived from different segments to bind sperm binding and maintain their motility in vitro. In experiment 1, the number of sperm attached to OEC derived from isthmus or ampulla, the motility of unattached sperm during co-culture and fertilizing ability were assessed. In experiment 2, heparin treated sperm (hsp) or no treated sperm (nsp) were used to evaluate OEC binding ability of capacitated sperm. In experiment 1, regardless of their origin, approximately 65% of the sperm were attached to OEC within 2h. From 6h of co-culture, the numbers of unattached sperm on ampullary OEC were significantly higher than those on isthmic OEC (p<0.005). From 12h of co-culture, the motility of unattached sperm on isthmic OEC were significantly higher than those on ampullary OEC(p<0.05). The cleavage rate of oocytes inseminated on OEC derived from isthmic segment was also significantly higher than those from ampullary segment (p<0.01). In experiment 2, the numbers of unattached hsp on OEC were significantly higher than those of controls (p<0.01), between 2-24h examination. From 12h of co-culture, the motility of unattached nsp were significantly greater than those of hsp (p<0.01). These results show that bovine OEC derived from the isthmus play more important role(s) for sperm binding, maintaining motility and fertilization in vitro than those from the ampulla, and heparin induced capacitation may change sperm binding ability on OEC in vitro.

• BMB Reports
• /
• 제33권2호
• /
• pp.97-102
• /
• 2000

Phosphoinositide-specific phospholipase C-${\gamma}1$ (PLC-${\gamma}1$) is a pivotal mediator in the signal transduction cascades induced by many growth factors. Using a yeast two-hybrid system, heat-shock protein 90 (Hsp90) was identified as a PLC-${\gamma}1$-binding protein. A co-immunoprecipitation experiment, using anti-PLC-${\gamma}1$ antibody, demonstrated an in vivo interaction between Hsp90 and PLC-${\gamma}1$ in the NIH-3T3 cells. The interaction in NIH-3T3 was unaffected by the PDGF treatment, inducing phosphorylation and activation of PLC-${\gamma}1$. Direct interaction between Hsp90 and PLC-${\gamma}1$ was confirmed by in vitro binding experiments using purified Hsp90 and PLC-${\gamma}1$. Furthermore, Hsp90 increased the $PIP_2$-hydrolyzing activity of PLC-${\gamma}1$ up to 2-fold at $0.1{\mu}M$ in vitro. Taken together, we show for the first time, the interaction of PLC-${\gamma}1$ with Hsp90, both in vivo and in vitro. We suggest that Hsp90 may play a role in PLC-${\gamma}1$-mediated signal transduction.

• Asian-Australasian Journal of Animal Sciences
• /
• 제15권7호
• /
• pp.1051-1056
• /
• 2002

In vitro binding efficacy of esterified glucomannan (E-GM) (0.1%) on aflatoxin B1 (AF) (300 ppb), ochratoxin A (OA) (2 ppm) and T-2 toxin (T-2) (3 ppm), when present alone or in combination, was evaluated in toxin-contaminated feed at pH 4.5 and 6.5. Esterified glucomannan showed significantly (p<0.01) higher binding with AF (81.6%), whereas those recorded with T-2 (27.8%) and OA (25.6%) were moderate. Binding of each toxin decreased as the number of toxins in feed increased. pH of medium showed no effect on mycotoxin binding ability of E-GM. A $2{\times}2{\times}2{\times}2$ factorial experiment of 5 week duration was conducted to study the effects of two dietary levels each of AF (0 and 300 ppb), OA (0 and 2 ppm), T-2 (0 and 3 ppm ) and E-GM (0 and 0.1%) on the immune competence of a total of 960 day-old commercial broilers. Reductions in size of thymus (by AF and T-2) and bursa (by AF) and antibody titers against Newcastle disease and Infectious Bursal disease (by all the toxins) were noted. Additive and antagonistic interactions were seen among the toxins on certain parameters. Esterified glucomannan significantly (p<0.01) improved antibody titers and weights of bursa ofFabricius and thymus indicating its counteracting efficacy against immunosuppression in mycotoxicosis of multiple origin.

• 한국유가공학회:학술대회논문집
• /
• 한국유가공기술과학회 2002년도 제54회 춘계심포지움 - 우유와 국민건강
• /
• pp.37-42
• /
• 2002

Lactoferrin is an iron-binding glycoprotein and its bacteriostatic and bactericidal effects on Gram-positive and Gram-negative bacteria have been well-known. However, certain kind of lactic acid bacteria are resistant against its antibacterial effects. Moreover, it is reported that lactoferrin promotes the growth of bifidobacteria by in vitro and in vivo experiments. In this experiment, lactoferrin-binding protein was found both in the membrane and cytosolic franctions of Bifidobacterium. Bifidobacterium was grown in anaerobic conditions in MRS broth containing cysteine, gathered by centrifugation and processed by sonication. The lactoferrin-binding proteins on the PVDF-membrane transferred after SDS-PAGE were detected by far-western method using biotinylated lactoferrin and streptavidin-labeled horse radish peroxidase. Observation in growth effects of lactoferrin on Bifidobacterium suggested that there is a relation between the presence of lactoferrin-binding proteins on the cells and their growth.

• 한국수정란이식학회지
• /
• 제29권2호
• /
• pp.127-132
• /
• 2014

This study was carried out to investigate the effects of tissue inhibitor of matalloproteinase-1 (TIMP-1), Activin A and Heparin binding epidermal growth factor (HB-EGF) on in vitro production of bovine embryos. In experiment 1, presumptive zygotes were cultured in the medium supplemented with TIMP-1 ($0.5{\mu}g/ml$), Activin A (100 ng/ml), or HB-EGF (100 ng/ml) at $39^{\circ}C$ in a humidified atmosphere of 5% (v/v) $CO_2$, 5% (v/v) $O_2$ and 90% (v/v) $N_2$. In experiment 2, TIMP-1 + HB-EGF or Activin A + HB-EGF combinations were supplemented in the culture medium. The developmental rate to blastocysts, hatching rate and total cell numbers of the blastocysts were evaluated in both experiments. The embryos cultured in medium without growth factor supplementation was used as control group. In experiment 1, the embryos cultured in medium supplemented with TIMP-1 and Activin A showed significantly higher developmental rate to blastocysts than those cultured with HB-EGF and control (36.9%, 34.1%, 21.2% and 23.1%, respectively) (P<0.0001). However, the hatching rate of blastocyst was significantly higher in embryos with HB-EGF than those with TIMP-1, Actvin A and Control groups (84.4%, 58.8%, 51.4% and 49.3%, respectively) (P<0.001). Total cell number per blastocyst was also significantly higher in embryos with HB-EGF group ($174.3{\pm}2.5$) than those with TIMP-1, Activin A (149.7 and 150.0, respectively) (P<0.05) and Control (119.0) (P<0.001). In experiment 2, embryos cultured with combined treatment of Activin A and HB-EGF resulted in significantly higher rates of blastocysts formation (48.0%), hatching rate (89.7%) and total cell number in blastocyst ($182.3{\pm}2.1$) than those with TIMP-1 and HB-EGF combination group (32.0%, P<0.001; 76.6%, P<0.05; $165.7{\pm}4.2$, P<0.001, respectively). Our data demonstrate that in vitro production of bovine embryos could be improved by combined supplementation of Activin A and HB-EGF in culture medium.

• BMB Reports
• /
• 제34권2호
• /
• pp.102-108
• /
• 2001

A time-dependent folding process was used to determine whether or not protein disulfide isomerase (PDI) plays an important role in the maturation of nascent lactoferrin polypeptides. Interaction between lactoferrin and PDI was analyzed according to the co-immunoprecipitation of the two proteins. The results indicate that lactoferrin folding requires a significant interaction with PDI and its binding is relatively brief compared to other nascent polypeptides. The amount of lactoferrin interacting with PDI increases up to half a minute and sharply decreases beyond this time point. During the refolding process that follows reduction by DTT, lactoferrin polypeptides heavily interact with PDI and the interaction period was extended compared to the normal folding process. In terms of the temperature effect on PDI-lactoferrin interaction, PDI binds to lactoferrin polypeptides longer at a lower temperature (here, $25^{\circ}C$) than $37^{\circ}C$. The lactoferrin-PDI interaction was also studied in vitro. According to the in vitro experiment data, PDI was still functional in cell lysates assisting lactoferrin folding into the mature form. PDI interacts with lactoferrin polypeptides for an extended period during the folding in vitro. During the refolding process in vitro, intermolecular aggregates and refolding oligomers matured into a functional form after PDI binds to the lactoferrin. These results suggest that PDI provides a prolonged chaperoning activity in the refolding processes and that there appears to be a greater requirement for PDI chaperone activity in the refolding of lactoferrin polypeptides.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2001년도 춘계학술발표대회
• /
• pp.13-13
• /
• 2001

This study was undertaken to evaluate the effects of catalase using xanthine (X) - xanthine oxidase (XO) system on in vitro maturation and fertilization in pig. When follicular oocytes were cultured in maturation medium with X and/or XO, the maturation rates were not significantly different between in medium with and without catalase despite of different culture periods. However, significantly (P<0.05) higher maturation rates were obrained in culture with X-XO system. The rates of degenerated oocytes were increased with culture periods prolonged, and were significantly (P<0.05) higher in medium without than with catalase at 120 h of culture. On the other hand, the parthenogenetic oocytes were observed with high proportions at 72 h of culture, hut were not different in medium with and without catalase at various times of culture. In another experiment, the frozen-thawed boar spermatozoa treated with X-XO system for in vitro fertilization. The penetration rates were higher in medium with that than without catalase during the in vitro fertilization with, none (P<0.05), XO and X＋XO. On the other hand, when sperm were treated with none, X, XO and X＋XO, lipid peroxidation were higher in medium without that than with catalase. However, the changes in sperm penetration and lipid peroxidation showed opposite patterns. The sperm suspensions were also treated with X and/or XO for assay of sulfhydryl (-SH) group content. Under the above all conditions, sperm-SH group were higher detected In medium with that than without catalase. The activity of sperm binding to zona pellucida was also evaluated through binding to salt-stored porcine oocytes. In control group, sperm binding to zona pellucida were higher than in medium with X, XO and X＋XO groups. No significant differences, however, were observed between medium with and without catalase. In conclusion, the exposure of follicular oocytes and spermatozoa to X-XO system may be caused stimulating in vitro maturation and fertilization in pig. This work was supported by grant No. 2000-1-22200-001-3 from the Basic Research Program of the Korea Science & Engineering Foundation.

• 생명과학회지
• /
• 제15권6호
• /
• pp.928-934
• /
• 2005

ATF2는 c-Fos와 c-Jun과 같은 전사인자이며 이들은 루신지퍼 단백질이다. 루신지퍼 단백질은 동형이합체 혹은 이형이합체를 형성하며 promoter 영역에 결합하여 전사조절에 중요한 역할을 한다. 세포의 전사인자 ATF2는 ATF/CRE site에 결합하며 특히 선택적인 interfamily 이형이량체를 형성함으로써 전사 조절에 있어서 다양한 메카니즘을 제공할 수 있다. 본 연구에서는 ATF2 cDNA를 6xHis를 가진 expression vector에 subcloning하여 E.cnli BL2l에서 발현시켰다. 6xHis tag은 nickel-chelating chromatography를 가능하게 하였다 발현된 ATF2는 In vitro binding pull-down assay에서 동형이합체를 이를 뿐만 아니라 AP-1 그룹의 인자들과 선택적인 이형이합체를 형성함을 보여 주었다. BATF와는 강하게 결합하였으며 c-Jun과도 안정된 이합체를 형성하였다. 그러나 c-Fos와는 이합체를 형성하지 않으므로서 AP-1그룹 내에서도 이합체 형성이 선택적으로 이루어짐을 알 수 있다.

• 한국가축번식학회지
• /
• 제27권2호
• /
• pp.153-161
• /
• 2003

본 연구는 돼지 동결-융해 정자의 배양에 의한 체외수정능력과 glycosidase activity를 평가하기 위하여 chlortetracycfine fluorescence분석법을 이용하여, glycosidase가 체외수정능력과 정자침입에 미치는 영향에 대하여 검토하였다. 또한 돼지난자의 투명대내에서 발견된 당잔기에 대한 정자의 glyco-sidase 특이성을 확인하기 위하여 $\alpha$-L-fucosidase, $\alpha$-D-mannosidase, $\beta$-D-galactosidase 및 N-acetyl-$\beta$-D-glucosaminidase ($\beta$-GlcNAc'ase)의 activity를 분석하였다. 그 결과 glycosidase activity는 동결정자의 응해 후 배양하지 않았을 때보다 2시간 배양했을 때 더 높게 나타났다. $\beta$-GlcNAc'ase의 activity 는 정자 배양 유무에 관계없이 다른 glycosidase 처리시보다 최소한 2배 이상 높게 나타났다. 또한 첨체반응이 유기된 정자의 비율은 glycosidase ($\alpha$-D-mannosidase; P<0.05)에 의해 영향을 받았으며 자를 배양하지 않은 경우보다는 배양된 정자에서 높게 나타났다. 그러나 배양시간에 따른 정자의 존성에 대해 glycosidase의 종류에 따른 유의차는 인정되지 않았다. 한편 투명대내 정자의 접착과 입에 대한 또 다른 실험에서, 서로 다른 glyco-sidase가 첨가된 배양액내에서 수정된 정자가 배양시간이 길어짐에 따라 정자의 침입율은 낮아졌다 $\beta$-GlcNAc'ase; P<0.05). 투명대내의 정자접착정도는 glycosidase의 첨가시에 무첨가시보다 접착정도가 더 높았으며, 가장 높은 접착율은 $\beta$-GlcNAc'ase 첨가시 나타났다. 또한 모든 glycosidase 처리시 2시간 배양한 정자보다는 배양하지 않은 정자에서 투명대에 대한 접착정도가 높게 나타났으며, $\alpha$-D-mannosidase의 처리시 유의적인 차이를 보였다 (P<0.05). 본 연구의 결과, $\beta$-GlcNAc'ase가 주로 돼지정자의 원형질막내에 존재하는 것으로 추측되며, 배양된 정자에 의한 투명대 접착정도와 침입율이 낮았음에도 불구하고 glycosidase activity가 증가하는 것으로 나타났다.

• 한국가축번식학회지
• /
• 제25권4호
• /
• pp.317-325
• /
• 2001

본 연구는 lipid peroxides를 생산하는 것으로 알려진 ascorbic acid (Asc; 0.5 mM)와 ferrous sulfate (Fe$^{2+}$; 1 mM)가 돼지 동결-융해 정자의 체외수정능력에 미치는 영향을 검토하였다. 그 결과, 동결정액의 융해 처리시 배양액내 Asc/Fe$^{2+}$ (38%)의 동시첨가시 대조구 (27%)에 비해 정자의 첨체반응이 유의적으로 높게 유기되었다 (P＜0.05). 정자침입율 또한 Asc/Fe$^{2+}$ (76%)를 동시에 첨가하므로서 대조구 (55%)에 비하여 유의적으로 높게 나타났다 (P＜0.05). 한편 정자의 peroxidation은 상기의 처리구에서 maiondialdehyde (MDA)의 생성에 기초를 두고 평가하였는데, 정자의 처리시 Asc/Fe$^{2+}$의 동시첨가에 의해 MDA의 생성이 증가하였으나 처리구 사이에서 유의적인 차이는 인정되지 않았다. 또한 sulfhydry1(-SH) group의 용량을 측정한 결과 Asc/Fe$^{2+}$를 동시에 첨가한 실험구에서 가장 높게 나타났으나 타처리구와의 사이에서 유의적인 차이는 인정되지 않았다. 또 다른 연구에서 동결-응해정자가 난자의 투명대에 접착하는 정도를 평가한 결과, 대조구와 Asc처리시 Fe$^{2+}$ 처리에 비하여 유의적으로 (P ＜ 0.05) 높은 정자의 접착을 나타냈으나 Asc/Fe$^{2+}$의 동시 처리에 의한 정자접착의 증가는 인정되지 않았다. 결과적으로 lipid peroxidation의 증가를 야기시키는 Asc/Fe$^{2+}$의 동시첨가는 투명대에서 정자의 접착능력을 증가시키지 않았음에도 불구하고 첨체반응과 침입능력을 향상시키는 것으로 나타났다.