• 한국동물학회지
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• 제31권1호
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• pp.21-28
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• 1988

dbcAMP에 의해 성숙이 억제된 생쥐난자의 수정능을 조사하기 위해 본 실험을 행하였다. dbcAMP로 일시 성숙이 억제되었던 난자를 배양액내에서 정자와 섞고 24시간 배양한 후 발생한 2세포기의 배아형성, 정자의 관입, 전액형성을 조사하여 2세포기로 배아발생이 진척된 것을 수정율의 기준으로 정하였다. dbcAMP를 처리하지 않은 난자는 약 53.3%의 수정율을 보였으며,dbcAMP가 함유되 배양액에서 배양한 후 기본 배양액에서 성숙시킨 난자들의 수정율은 dbcAMP의 처리시간에 비례하여 낮으나, 정자의 관입은 정상적으로 일어났다. 전자현미경적 관찰에 의하면 dbcAMP 처리는 난자의 미세구조에 어떠한 변화도 야기하지 않았다. 따라서 dbcAMP를 사전처리하여 성숙을 억제한 난자라할지라도 어느 정도의 수정능력을 보유하고 있다고 사료된다.

• 한국수정란이식학회지
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• 제26권4호
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• pp.251-256
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• 2011

Presently, the effect of 0.5 mM dibutyryl cAMP (dbcAMP)-supplemented maturation medium during different incubation time on meiotic arrest (germinal vesicle) and resumption (metaphase II) of porcine oocytes and embryonic development of porcine oocytes following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Porcine cumulus oocyte complexes (COCs) were cultured in 0.5 mM dbcAMP for 17, 22, 27, or 42 h, and an additional 22 h without 0.5 mM dbcAMP. The nuclear status was examined at each time point. Oocytes cultured from 39~49 h displayed more than 80% meiotic resumption. More than 85 % of meiotic arrest was presented at 17~22 h. Oocytes were cultured for 22 h with 0.5 mM dbcAMP and additional 22 h without dbcAMP to assess developmental potential following IVF or PA. There were no significant differences in blastocyst rates among the dbcAMPIVF, IVF, dbcAMP-PA, and PA groups, although cleavage rate of IVF group was significantly higher than those of dbcAMP-PA, and PA groups. In conclusion, 0.5 mM dbcAMP influenced meiotic maturation of porcine oocytes depending on incubation time of oocyte, although embryonic development was not improved in both IVF and PA.

• 한국동물학회지
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• 제18권1호
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• pp.19-26
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• 1975

自記放射法을 이용하여 dbcAMP와 theophylline이 未成熟卵子의 RNA合成에 미치는 영향을 관찰하였다. 培養中인 未成熟卵子의 RNA合成은 dbcAMP와 theophylline에 의하여 抑制를 받았다. dbcAMP나 theophylline은 培養液(modified Krebs-Ringer bicarbonate solution)內에 100 $\\mu$g/ml 정도 들어 있으며 핵막(germinal vesicle)의 붕괴 되지 못하고 그대로 存在하며 그동안의 RNA合成은 극히 억제된 채로 남아 있다. 그러나 培養을 시작하여 2$\\sim$3時間後, 즉 핵막붕괴가 끝난 다음에 이들 억제물질을 배양액에 添加하면 正常卵子와 같이 성숙분열이나 RNA合成이 억제 됨이 없이 진행된다. 24時間동안 dbcAMP나 theophylline으로 성숙이 억제 되었던 卵子도 이들 억제물질을 제거하면 즉시 成熟分裂이 진행되며 RNA合成도 正常的으로 일어난다. 이런 結果로 미루어 dbcAMP등의 RNA合成 抑制機作에 한 가지 가능성을 추측할 수 있다. 즉 dbcAMP나 theophylline의 처리에 의해 細胞質內 cAMP의 농도가 높아지고 이 cAMP는 핵막붕괴나 染色質의 응집에 관여하는 단백질 合成을 誘導할 mRNA合成을 억제하며 이 때문에 卵子는 핵을 보유한채 그대로 남아 있는 것이다.

• 약학회지
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• 제51권1호
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• pp.35-43
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• 2007

To investigate the immunomodulatory roles of cyclic AMP (CAMP) on macrophage- and T lymphocyte-mediated immune responses, CAMP elevating agents were employed and carefully re-examined under the activation conditions of the cells. Various inhibitors tested dose-dependently blocked tumor necrosis factor (TNF)-${\alpha}$ production with IC$_{50}$ values ranged from 0.04 to 300 ${\mu}$M. Of the inhibitors, cAMP-elevating agents showed lower cytotoxicity assessed by lactate dehydrogenase (LDH) release, suggesting less toxic and more selective. In particular co-treatment of dbcAMP with a protein kinase C inhibitor staurosporine displayed the synergistic inhibition of TNF-${\alpha}$ production. The modulatory effect of dbcAMP on TNF-${\alpha}$ and nitric oxide (NO) was significantly affected by treatment time of dbcAMP. Thus, post-treatment of dbcAMP (three hours before LPS) abrogated dbcAMP's inhibitory activity and rather enhanced TNF-${\alpha}$ level up to 60%. In contrast, additional NO production was shown at the co-treatment of dbcAMP with LPS. Unlike simultaneous treatment of phorbol 12-myristate 13-acetate (PMA) and interferon (IFN)-${\gamma}$co-treatment, the combination of dbcAMP with other NO-inducing stimuli did not show drastic overproduction of NO. cAMP elevating agents also diminished splenocyte proliferation stimulated by concanavalin (Con) A, phytohemaglutinin A (PHA) and lipopolysaccharide (LPS). In addition, dbcAMP but not rolipram strongly suppressed CD8$^+$ T cells (CTLL-2). Finally, cAMP elevating agents were differentially involved in regulating CD98-mediated cell-cell adhesion. Thus, dbcAMP and rolipram significantly enhanced the cell-cell adhesion, whereas forskolin blocked. Therefore, our results suggest that CAMP elevating agents participate in various immune responses mediated by macrophages and T cells with a different fashion depending on cellular environments and activation signals.

• 한국동물학회지
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• 제28권2호
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• pp.99-107
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• 1985

생쥐난자의 성숙을 억제하는 것으로 알려진 dbcAMP와 progesterone을 동시에 처리하였을 때의 영향을 알아보기 위하여 본 실험을 행한 결과는 다음과 같다. 1) 10 $\\muM$/ml의 dbcAMP 혹은 2 $\\muM$/ml의 progesterone을 단독처리하였을 때에는 난자의 성숙을 억제하지 못했다. 2) 그러나 dbcAMP(10 $\\mug$/ml)와 progesterone(2 \\mug$/ml)을 동시에 배양액에 처리하였을 때에는 난자의 성숙이 억제되었다. 3) 난자를 먼저 4시간동안 위의 두 억제제가 들어있는 배양액에서 배양한 뒤 다시 정상배양액에 옮겨 계속 배양하면 나자들은 대조군에서와 같은 비율로 성숙을 하였다. 위의 결과로 보아 dbcAMP와 progesterone이 동시에 배양액에 존재할 때에는 각각 단독 존재하였을 때 영향이 없는 농도의 dbcAMP와 progesterone으로도 능히 난자의 성숙을 억제 시킬 수 있었으며 이 같은 억제효과는 가역적임을 알게되었다.

• 한국동물학회지
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• 제30권1호
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• pp.89-98
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• 1987

생쥐 미성숙 난자를 재료로 하여 난자 성숙 억제제인 dbcAMP 존재하에서 GVBD 난자와의 융합에 따른 체외성숙 양상을 조사하였다. 기본 배양액 내에서 3시간이 경과하였을 때 GVBD 난자와 융합된 미성숙 난자 뿐만이 아니라 미성숙 난자 뿐만이 아니라 미성숙 난자끼리 융합된 것들도 모두 성숙을 재개하였다. 그러나 dbcAMP가 함유된 배양액 내에서 3시간 경과 하였을 때는, 미성숙 난자끼리 융합된 것들은 모두가 GV상태로 성숙이 억제된 채로 있었고 반면에 GVBD 난자와 융합된 미성숙 난자들은 비록 dbcAMP가 존재하더라도 보두 성숙을 재개하였다. dbcAMP가 함유된 배양액 내에서 20시간이 경과하였을 때 미성숙 난자끼리 융합된 것들은 여전히 성숙이 억제되어 있었으나 GVBD 난자와 융합된 미성숙 난자들은, 기본 배양액 내에서 배양된 융합 난자들과 마찬가지로 다수가 하나 혹은 두개의 극체를 형성하는 제2 감수분열 중기에 진입하였다. 두 개의 극체를 방출한 융합난자들 중에는 하나의 세포질 내에 감수분열 중기방추사가 두 곳에서 형성되는 것이 관찰되었다. 이 실험 결과로 보아 이미 성숙이 재개된 난자내에는 난자 성숙 억제제인 dbcAMP의 억제효과보다 더 영향이 큰 난자 성숙 유도 물질이 있으며, 이 물질이 융합하고 있는 미성숙 난자내로 이전하여 dbcAMP에 의해서 그 성숙이 억제된 미성숙 난자의 성숙을 유도한다는 것을 알 수 있다.

• 한국동물학회지
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• 제18권1호
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• pp.27-40
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• 1975

dbcAMP와 theophylline이 난자의 성숙을 억제하는 기작과 난자내 glycogen 함량과의 관계를 밝혀보기 위하여 실험한 결과는 다음과 같다. 1. 생쥐 여포난자의 PAS 양성물빌은 glycogen이며 핵의 성숙분열이 진행됨에 따라 glycogen의 함량은 감소한다. 2. dbcAMP나 theophylline에 의해 핵의 성숙이 억제된다 하더라도 난자내의 glycogenolysiss는 촉진된다. 이에 반해 핵분열이 일어나지 않은 난자는 배양이 진행되더라도 glycogen을 그대로 유지하고 있다. 일단 dbcAMP나 theophylline에 의해 성숙이 억제되었던 난자가 성숙과정에 들어가려면 다시 glyconeogenesis가 일어나 세포질내의 glycogen의 양이 회복하며 이때의 glycogen은 핵성숙과 더불어 소모된다. 3. Glycogen의 회복은 배양액내의 glucose 유무에 관계없이 이루어지며 따라서 이는 난자내 glucose 혹은 다른 전구물질에 의해 이루어지리라고 추측된다. 4. 결국 dbcAMP나 theophylline은 난자내 cAMP의 양을 증가시켜 glycogen의 소모를 일으키는 것으로 생각되며, glycogenolysis와 난자의 핵 성숙과정이 별개로 진행되기는 하지만 성숙의 요건은 일정량 이상의 glycogen이 함유되어 있어야 한다는 것을 알게되었다.

• Applied Microscopy
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• 제7권1호
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• pp.21-43
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• 1977

This experiment was undertaken in order to localize the labeled dbcAMP (dibutyryl cyclic AMP) in oocytes whose development has been suppressed by cold dbcAMP for 6 or 19 hours in vitro. Mouse oocytes were obtained from the ovaries of 3-4 week old A strain female mice, by puncturing the Graafian follicles in the modified Krebs-Ringer bicarbonate salt solution under the dissecting microscope. Those oocytes which have intact germinal vesicle were cultured in the basic culture medium supplemented with 0.4% bovine serum albumin (BSA). Cultivation of the oocytes was carried out in a microtube developed by Cho (1974). The cultures were then incubated in a humidified 5% $CO_2$ incubator maintained at $37^{\circ}C$ for 6 or 19 hours (Donahue, 1968). DbcAMP was added to culture medium for a final concentration of 100ug/ml, and $^3H-dbc$ AMP (specific activity 13 Ci/mM) for a final concentration of $40{\mu}Ci/ml$ was also added to the medium. For electron microscopic autoradiography, those oocytes recovered from the culture were washed with phosphate buffer (pH 7.4), and immediately prefixed in a 2.5% glutaraldehyde overnight and postfixed for 2 hours at $4 ^{\circ}C$ in 1% osmium tetroxide in phosphate buffer with pH 7.4 (Palade, 1952). After fixation, the materials were dehydrated in graded alcohol series and embedded in Epon 812 mixture based on the standard procedures (Luft, 1961). The thin sections $600-700{\AA}$ thick were mounted on the grids of 200 meshes. The grids containing sections were coated with a nuclear emulsion Kodak NTB-3 and stored in a cold dark box (at $4^{\circ}C$) for 3 weeks. After exposure, the samples were developed with Kodak D-19 and stained with uranyl acetate and lead citrate. Routine observation was made with Hitachi HU-11E electron microsocope. The results of the observation were as followings: 1. It was found that the labeled dbcAMP penetrated the egg plasma membrane and dispersed at random in the cytoplasm. 2. It was also observed that most of the labeled dbcAMP was attached to microfibrillar lattices portion of the oocyte cytoplasm. There fore, it is presumed that the receptor of the dbcAMP is localized in the microfibrillar lattices of the oocyte. 3. It also seems that some other cell organells such as mitochondria, Golgi complex, cortical granules are not directly related to the action of the dbcAMP. 4. The labeled dbcAMP was neither observed in the membrane nor in the nucleus. Therefore, it seems that there is no relationship between the concentration of dbcAMP and the nuclear membranous permeability. 5. There was no difference in number of dbcAMP particles when oocytes were cultured for 6 hours and 19 hours. 6. However, it was observed that, in same of the oocytes suppressed in germinal vesicle by dbcAMP for 19 hours, cell organells were moved and concentrated to a small portion of the cytoplasm, and that the morphology of the organells greatly changed to an abnormal. form. Therefore, it is supposed that those oocytes were in the process of degeneration. From the above results, it is expected that dbcAMP penetrated the egg membrane and was bound to the receptor which seems to be located in the microfibrillar lattiees portion, and that this dbcAMP-receptor complex inhibited some enzyme system of the oocytes which are essential for the germinal vesicle breakdown.

• 약학회지
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• 제43권3호
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• pp.378-384
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• 1999

Retinoic acid (RA) and dibutyryl cyclic AMP (dbcAMP) induce the differentiation of the multipotent embryonic carcinoma cell line, F9 cells, into parietal endoderm like cell. The F9 cells are highly proliferative doubling approximately 12 hourse. S Phase is predominant, lasting 10 hours and G2/M phase occupies most of the remaining cycle (2 hours) and G1 phase is nearly non-existent. In this study, we showed the effect of RA and dbcAMPon the cell cycle associated molecules (especially around G1 phase) during F9 cell differentiation. Differentiation of F9 cells was induced by the combined addition of RA ($10^{-7}M$) and dbcAMP (0.5mM), and cells were harvested daily up to 4 days. Flow cytometric analysis showed the prolongation of G1 phase around 30 hours after induction. Western blot analysis revealed that the amount of cyclin D1 and cdk2 were increased at day 4. However, histone H1 kinase activity of cdk2 was decreased. These data strongly suggest that RA and dbcAMP induce the growth arrest of F9 cells at G1 phase by decreasing the activity of cdk2, although they have increased the protein contents of cyclin D1 and cdk2. The reason for the discrepancy between the H1 kinase activity and protein contents are not clear yet.

• 한국동물위생학회지
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• 제31권4호
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• pp.449-456
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• 2008

The differentiation of mouse embryonic stem(ES) cell into smooth muscle cells(SMC) may play a major role in cardiovascular development and under pathophysiological conditions. Therefore, in the present study, we have examined the differentiation of ES cells and its related gene expression. SMC differentiation was indicated by cellular morphology and time-dependent induction of dibutyryl adenosine 3,5-cyclic monophosphate(DBcAMP)and retinoic acid(RA) on smooth muscle ${\alpha}$-actin($SM{\alpha}A$), smooth muscle myosin heavy chain(SMMHC) gene expression. The control was undifferentiated ES cells(protein expressions represent 50-60kDaOct-4). The results of this study show that morphology of embryoid body and confirmation of $SM{\alpha}A$ expression by immunocytochemistry. Moreover, SMMHC and desmin expression was significantly increased by time dependent manner(5, 7, 15 days), in contrast to $SM{\alpha}A$ expression was slightly decreased on 15days. In conclusion, DBcAMP and RA stimulate mouse ES cells differentiation into SMC and enhanced $SM{\alpha}A$, SMMHC and desmin expression.