• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1994.04a
• /
• pp.258-258
• /
• 1994

본 연구에서는 토양균이 생성하는 2차 대사산물로부터 세포증식 촉진인자 (Maturation Promoting Factor. MPF)인 cdc2/cdk2-cyclin의 복합체를 특이적으로 억제하는 물질을 분리하고 그 물질의 생리활성을 조사하였다. 토양으로부터 순수분리된 300여개의 토양균에서 생성된 배양액을 취하여 MPF의 특이적인 기질인 합성 peptide (CSH103 :HATPPKKKRK)를 사용하여 인산화 활성을 측정하였다. 그중 MPF 활성 억제능이 90％ 이상인 19개의 균주를 1차적으로 선정한 후 각각에 대하여 열/pH에 대한 안정성, 각종 용매에 대한 추출성 등 이화학적 성질을 규명하였으며 이중 균주 LPL 931로부터 MPF 활성 억제제를 분리하고자 하였다. 예비실험의 결과로부터 토양균 LPL 931을 대량배양하여 열처리하고, 비이온성 수지인Amberlite XAD-2에 결합시키고 70% acetone으로 용출시켰다. 이 추출물로부터 ethylacetate와 n-butanol을 사용하여 MPF 억제 활성물질을 추출하였다. 이 추출액을 실리카겔 관 크로마토그래피, 분취 TLC, 분취 HPL를 하여 MPF 활성 억제 분획을 분리하였다.

• Reproductive and Developmental Biology
• /
• v.36 no.4
• /
• pp.261-267
• /
• 2012

The technique of SCNT is now well established but still remains inefficient. The in vitro development of SCNT embryos is dependent upon numerous factors including the recipient cytoplast and karyoplast. Above all, the metaphase of the second meiotic division (MII) oocytes have typically become the recipient of choice. Generally high level of MPF present in MII oocytes induces the transferred nucleus to enter mitotic division precociously and causes NEBD and PCC, which may be the critical role for nuclear reprogramming. In the present study we investigated the in vitro development and pregnancy of White-Hanwoo SCNT embryos treated with caffeine (a protein kinase phosphatase inhibitor). As results, the treatment of 10 mM caffeine for 6 h significantly increased MPF activity in bovine oocytes but does not affect the developmental competence to the blastocyst stage in bovine SCNT embryos. However, a significant increase in the mean cell number of blastocysts and the frequency of pregnant on 150 days of White-Hanwoo SCNT embryos produced using caffeine treated cytoplasts was observed. These results indicated that the recipient cytoplast treated with caffeine for a short period prior to reconstruction of SCNT embryos is able to increase the frequency of pregnancy in cow.

• The Korean Journal of Zoology
• /
• v.30 no.1
• /
• pp.89-98
• /
• 1987

The research of fused oocytes was conducted to investigate the in vitro mejotic maturation of immature oocytes (GV oocytes) fused with oocytes in germinal vesicle breakdown (GVBD oocytes) in the presence of dbcAMP which is known as one of the strong inhibitors to GVBD. The immature oocytes fused together as well as those fused with GVBD oocytes proceeded to GVBD in 3 hr culture in plain medium. But in the medium containing dbcAMP (100$\mu$g/ml), the immature oocytes fused together did not show any GVBD and thus the fusion itself could not affect the inhibitory activity of dbcAMP. However, all of the immature oocytes fused with GVBD oocytes underwent GVBD in 3 hr culture despite of the presence of dbcAMP. When the culture was extended to 20 hr, nearly all of the immature oocytes fused together were still arrested at the GV stage in the presence of dbcAMP. But most of the fused oocytes which had shown GVBD during 3 hr culture developed to metaphase II stage extruding one or two polar bodies regardless of the presence of dbcAMP. In this experiment, it was found that two sets of the metaphase chromosomes were somewhat concomitant with a pair of the polar bodies in the fused egg. Upon the results of the present studies, it is assumed that there may be a maturation promoting factor(s) in the cytoplasm of the GVBD occytes, and this factor(s) possibly nullifies the function of dbcAMP.

• Molecules and Cells
• /
• v.44 no.9
• /
• pp.647-657
• /
• 2021

As a pancreatic inflammatory marker, regenerating islet-derived protein 3A (Reg3A) plays a key role in inflammation-associated pancreatic carcinogenesis by promoting cell proliferation, inhibiting apoptosis, and regulating cancer cell migration and invasion. This study aimed to reveal a novel immuno-regulatory mechanism by which Reg3A modulates tumour-promoting responses during pancreatic cancer (PC) progression. In an in vitro Transwell system that allowed the direct co-culture of human peripheral blood-derived dendritic cells (DCs) and Reg3A-overexpressing/ silenced human PC cells, PC cell-derived Reg3A was found to downregulate CD80, CD83 and CD86 expression on educated DCs, increase DC endocytic function, inhibit DC-induced T lymphocyte proliferation, reduce IL-12p70 production, and enhance IL-23 production by DCs. The positive effect of tumour-derived Reg3A-educated human DCs on PC progression was demonstrated in vivo by intraperitoneally transferring them into PC-implanted severe combined immunodeficiency (SCID) mice reconstituted with human T cells. A Reg3A-JAK2/STAT3 positive feedback loop was identified in DCs educated with Reg3A. In conclusion, as a tumour-derived factor, Reg3A acted to block the differentiation and maturation of the most important antigen-presenting cells, DCs, causing them to limit their potential anti-tumour responses, thus facilitating PC escape and progression.

• The Journal of Korean Obstetrics and Gynecology
• /
• v.24 no.2
• /
• pp.52-67
• /
• 2011

Objectives: This study was designed to investigate the effects of Jokyeongjongok -Tang(JJT) on the progression of the estradiol valerate(EV)-induced polycystic ovaries(PCO) in rats. Methods: PCO was induced by single intramuscular injection with estradiol valerate(EV)(4 mg) in female rats. Normal group(n=8) were injected with sesame oil and orally administrated distilled water for sixty days. PCO control group (n=8) were injected with EV and orally administrated distilled water for sixty days. JJT treated group(n=8) were injected with EV and orally administrated JJT for same duration. At the end day of experiment, we measured weights of body, ovaries, adrenal glands and uterus. The histopathological changes of ovaries were also evaluated. And we observed the NGF and CRF expression by immunohistochemistry. Results: The results were as follows - The weights(mg) of ovaries of JJT treated group($58.4{\pm}9.4$) were significantly increased(p<0.01) compared with PCO control group($42.3{\pm}8.5$). - The numbers of mature follicles of JJT treated group($10.1{\pm}2.5$) were significantly increased(p<0.01) compared with PCO control group($6.1{\pm}2.1$). - The numbers of cystic follicles of JJT treated group($1.8{\pm}1.4$) were significantly decreased(p<0.05) compared with PCO control group($3.8{\pm}1.5$). - The expressions of NGF-immunoreactive cells in the ovaries of JJT treated group were weaker than PCO control group. Conclusions: From the above results, we concluded that Jokyeongjongok-Tang (JJT) contributes to a normal maturation of follicles and has the effects of promoting a normal ovulation. And these effects may be related with the decreased NGF activities in the ovaries.

• Korean Journal of Animal Reproduction
• /
• v.17 no.2
• /
• pp.75-83
• /
• 1993

Purine has been identified in the preparation of follicular fluid and shown an activity in maintaining oocyte meiotic arrest. Therefore this study was performed to examine the inhibitory effect of purine on germinal vesicle break down(GVBD) in the presence and absence of human fetal cord serum(HFCS) or human mature follicular fluid(HMFF), as a protein source, in vitro culture. Immature oocytes(GV stage) were collected from ovaries of 21∼28 days old ICR mice by puncturing the antral follicles with a fine needle, at 48 hrs after PMSG injection. Some of the oocytes were denuded by drawing the cumulus-enclosed(complex) oocytes in and out of a pasteur pipet. Complex oocytes and denuded oocytes were cultured 3 hrs. in T6 media containing 0.75mM adenosine or/and 4mM hypoxanthine, with HFCS or HMFF. Their GVBD rates were observed at every 1 hr. during the culture time. Both adenosine and hypoxanthine have shown a time-dependent inhibitory effect on GVBD in complex and denuded oocytes and the inhibitory effect was maximized in culture medium containing hypoxanthine and adenosine. HFCS and HMFF increased the GVBD rates in the presence of the purines, thus HFCS and HMFF may contain a factor that could reverse the inhibitory effect of purines. Also complex oocytes were more sensitive to not only the inhibitory effect of purines but the promoting action of HMFF on GVBD than denuded oocytes. Therefore it was reconfirmed that granulosa cells play an important part in meiotic arrest and resumption.

• Maxillofacial Plastic and Reconstructive Surgery
• /
• v.21 no.1
• /
• pp.1-12
• /
• 1999

The purpose of this study was to evaluate the healing process of the calvarial defect filled with hydroxylapatite(HA) and $TGF-{\beta}$ in Rat. 72 Sprague-Dawly rats were divided into 3 groups, control and two experimental groups. Bony defect were artificially prepared in the calvaria of all 72 rats and followed by implantation of HA (experimental group of 24 rats) and HA+$TGF-{\beta}$(another experimental group of 24 rats) into the defects. Sequential sacrifice was performed at 1, 2, 4, 6, 8, 12 weeks of experiment. Obtained specimen was stained with Hematoxylin and Eosin, Masson's Trichrome and Immunohistochemistry. The results were as follows, 1. Granulation tissue was prominent on control group in 1 and 2 weeks. Bony defects were filled with dense fibrous tissue through the whole experimental period and osteoinduction could not be observed in all groups. 2. Inflammatory cell infiltration was prominent on control group in 1 and 2 weeks and osteoclastic activity was high in HA implanted experimental group at 1 and 2 weeks. 3. Inflammatory cell infiltration was less and maturation of fibrous tissue could be found on HA+$TGF-{\beta}$ implanted experimental group at 1 and 2 weeks. 4. Osteoconduction activity was high in HA+$TGF-{\beta}$ implanted experimental group at 2 and 4 weeks but there was no difference after 6 weeks among 3 groups. 5. In grafted site of HA+$TGF-{\beta}$ implanted group, osteonectin expression was slightly increased from 1 week to 6 weeks. In the host site, it was increased from 1 to 4weeks. 6. In grafted site of HA+$TGF-{\beta}$ implanted group, osteocalcin expression was high at 4 weeks. In the host site, we could find the difference among 3 groups. From above results, the HA with mixture of $TGF-{\beta}$ has the potentiality of promoting bone formation in the bony defect area in the rat.

• Animal Bioscience
• /
• v.35 no.7
• /
• pp.1010-1020
• /
• 2022

Objective: The experiment was conducted to evaluate the effects of maternal undernutrition during late pregnancy on the expressions of genes involved in growth and development in ovine fetal perirenal brown adipose tissue (BAT). Methods: Eighteen ewes with singleton fetuses were allocated to three groups at day 90 of pregnancy: restricted group 1 (RG1, 0.33 MJ metabolisable energy [ME]/kg body weight [BW]^0.75/d, n = 6), restricted group 2 (RG2, 0.18 MJ ME/kg BW^0.75/d, n = 6), and a control group (CG, ad libitum, 0.67 MJ ME/kg BW^0.75/d, n = 6). The fetuses were removed at day 140 of pregnancy. All data were analyzed by using the analysis of variance procedure. Results: The perirenal fat weight (p = 0.0077) and perirenal fat growth rate (p = 0.0074) were reduced in RG2 compared to CG. In fetal perirenal BAT, the protein level of uncoupling protein 1 (UCP1) (p = 0.0001) was lower in RG1 and RG2 compared with CG and UCP1 mRNA expression (p = 0.0265) was decreased in RG2. The protein level of myogenic factor 5 (Myf5) was also decreased in RG2 (p = 0.0001). In addition, mRNA expressions of CyclinA (p = 0.0109), CyclinB (p = 0.0019), CyclinD (p = 0.0015), cyclin-dependent kinase 1 (CDK1) (p = 0.0001), E2F transcription factor 1 (E2F1) (p = 0.0323), E2F4 (p = 0.0101), and E2F5 (p = 0.0018) were lower in RG1 and RG2. There were decreased protein expression of peroxisome proliferator-activated receptor-γ (PPARγ) (p = 0.0043) and mRNA expression of CCAAT/enhancer-binding protein-α (C/EBPα) (p = 0.0307) in RG2 and decreased PPARγ mRNA expression (p = 0.0008) and C/EBPα protein expression (p = 0.0015) in both RG2 and RG1. Furthermore, mRNA expression of bone morphogenetic protein 4 (BMP4) (p = 0.0083) and BMP7 (p = 0.0330) decreased in RG2 and peroxisome proliferator-activated receptor co-activator-1α (PGC-1α) reduced in RG2 and RG1. Conclusion: Our observations support that repression of regulatory factors promoting differentiation and development results in the inhibition of BAT maturation in fetal perirenal fat during late pregnancy with maternal undernutrition.

• Journal of Life Science
• /
• v.18 no.1
• /
• pp.114-119
• /
• 2008

The molecular machinery controlling cell cycle is centered around the regulation of the activity of maturation-promoting factor (MPF), a complex composed of a catalytic Cdc2 and the cyclinB regulatory subunit. Cdc2 kinase is inactivated by phosphorylation of inhibitory kinase, Wee1. It has been known that there are three different Wee1 kinases in the mammalian cell, Wee1A, Wee1B and Myt1. To investigate the regulatory mechanism of Wee1 kinases, the phosphorylation and degradation of Wee1A and Wee1B were checked in the Xenopus oocyte cell cycle. When Wee1 kinases were injected into frog oocyte, Wee1B was more stable than Wee1A. Wee1A and Wee1B kinase were phosphorylated by many kinases such as PKA and Akt. The roles of amino or carboxyl terminal in mouse Wee1A or Wee1B kinase were investigated using chimeric constructs. The degree of protein phosphorylation, degradation and cell cycle progression were different between chimeric constructs. The amino domain of Wee1A was implicated in the protein phosphorylation and degradation while amino domain of Wee1B and carboxyl domain of Wee1A were involved in the activity regulation. These results suggested that the domains of Wee1 kinase have different and significant roles in regulating the Wee1 kinases in the cell cycle progression.

• Journal of Embryo Transfer
• /
• v.16 no.3
• /
• pp.223-231
• /
• 2001

The success of nuclear transplantation with mammalian oocytes depends critically on the potential of oocytes activation, which mainly caused to prevent the re-accumulation of maturation promoting factor (MPF). This study was conducted to compare the effect of combined treatment of lonomycin with a Hl-histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on activation of bovine oocytes. In vitro matured bovine oocytes with the first polar body (PB) and dense cytoplasm were assigned to 3 experimental groups. For activation treatment, oocytcs were exposed to 5 $\mu$M lonomycin for 5 min (Group 1), and followed by 1.9 mM dimethylaminopurine (DMAP) for 3 h (Group 2) or followed by 2 mM sodium pyrophosphate (SPP) for 3 h (Group 3). The activation effects in the three treatments and the control group (untreated) were judged by the extrusion of the second PB and formation of a pronucleus (PN). Differences among groups were analysed using one-way ANOVA after arc-sine transformation of proportional data. All three treatments led to high activation rates (90% to 95%), with significant difference from the control. However, the extrusion of the second PB and the rate of PN formation differed remarkably among treatments. In Group I and 3, about 95% of the oocytes had extruded the second polar body, but one PN had formed in a higher proportion of oocytes in Group 3 than in Group 1 (90% vs. 5%). In experiment 2, the rates of cleavage and development into blastocysts in Group 1 were significantly lower than those of Group 2 and 3 (8.7% and 0% vs. 50.5% and 11.6%, and 44.6% and 7.2%, respectively, P<0.05). In experiment 3, ~80% of parthenotes in Group 1 were developed with haploid chromosomal sets. However, when ionomycin was followed immediately by DMAP (Group 2). only 20% of parthenotes were haploid. In Group 3, combined treatment with ionomycin and SPP, the appearance of abnormal chromosomal tracts was significantly (P〈0.05) reduced and the proportion of haploid parthenotes was increased to 85% (17/20) than in Group 2. These results demonstrate that SPP acted as a cdc2 kinase inhibitor and formed the haploidy in oocyte activation. Thus, the present study suggests that cdc2 kinase inhibitor, such as sodium pyrophosphate, may have an effective role in oocyte activation for the production of cloned embryos/animals by nuclear transplantation.