• Reproductive and Developmental Biology
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• 제31권3호
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• pp.139-143
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• 2007

본 연구는 체세포를 이용하여 생산된 복제 한우 수소의 번식능력을 검토하기 위해 실시하였다. 복제 한우 수소(C-38 및 C-39) 또는 일반 한우 종모우로부터 정액을 채취하여 정자의 수 및 동결 전 후의 생존성 등을 살펴보았으며, 정자의 운동성 등은 computer assisted sperm analysis(CASA)를 이용하여 측정하였다. 또한, 이들의 수정 능력을 확인하기 위하여 체외수정과 인공수정을 각각 실시하였다. 정액 성상에서는 복제 수소들과 일반 종모우 간에 정액의 양, 정자의 농도 및 동결융해 후의 생존성 등에서 차이가 나타나지 않았다. CASA를 이용한 분석에서 운동성, 곡선 운동 속도(VCL), 직선운동 속도(VSL) 및 평균 진행 속도(VAP) 등은 복제 수소의 정액이 일반 종모우의 정액에 비하여 유의적으로 높았다 (p<0.05).체외수정에 따른 수정란의 분화율 및 배반포로의 발달율은 복제 수소와 일반종모우 간에 차이가 나타나지 않았다. 복제소 정액(C-38)을 이용하여 인공수정을 한 5두의 체세포 복제 대리모에서 암 수 각각 한 두씩의 건강한 복제 후대 송아지 2두를 생산하였다. 이상의 결과를 종합하여 보면, 실험에 공시된 복제 수소 개체간의 차이가 나타나기는 하였지만, 복제 수소는 정액 성상과 정자의 운동성 등에서 일반 종모우와 차이가 없었으며, 또한 인공수정을 통해 송아지를 생산함으로써 정상적인 번식능력이 있음을 확인하였다.

• 생명과학회지
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• 제22권12호
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• pp.1621-1627
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• 2012

세포의 이동은 성장, 면역 작용, 그리고 혈관 신생 등 많은 생리현상에 중요한 역할을 한다. 또한 염증 및 종양 세포 침윤 등의 다양한 병리적 현상과도 밀접한 연관이 있다. 본 연구에서는 lysophosphatidic acid (LPA)는 활성산소의 생성을 통해 SKOV-3 난소암세포의 이동을 조절한다는 것을 관찰하였다. 먼저, 난소 암세포인 SKOV-3에서 LPA에 의한 세포의 이동이 강하게 일어남을 확인하였다. LPA에 의한 SKOV-3 세포의 이동은 phosphatidylinositol 3-kinase (PI3K)/Akt 신호전달체계를 저해시키는 약물에 의해서 완벽히 억제됨을 확인하였으나 ERK 신호전달체계를 저해시키는 약물에 의해서는 전혀 영향을 받지 않았다. 그리고 SKOV-3 세포에서 LPA에 의한 활성산소 형성이 시간에 따라 강하게 일어남을 확인하였다. 더욱이 LPA에 의한 활성산소 형성도 PI3K 또는 Akt의 저해제에 의해서 완벽히 억제됨을 확인하였으나 ERK 신호전달을 억제하였을 때는 거의 영향을 받지 않았다. SKOV-3 세포에서 LPA에 의해 생성된 활성산소는 diphenylene idonium (DPI, $10{\mu}M$), apocyanin (Apo, $10{\mu}M$)과 같은 NADPH oxidase 억제제를 전 처리하였을 때 활성산소가 형성되지 못함을 관찰하였다. 그러나 xanthine oxidase (allopurinol, Allo, $10{\mu}M$), cyclooxygenase (indomethacin, Indo, $10{\mu}M$), 또는 mitochondrial respiratory chain complex I (rotenone, Rot, $10{\mu}M$)를 억제하였을 때는 LPA에 의한 활성산소 형성에 영향을 주지 못함을 확인하였다. 마지막으로 활성산소 억제제인 N-acetylcysteine (NAC, $10{\mu}M$)에 의해서 LPA에 의한 암세포의 이동이 억제됨을 관찰하였다. 이와 더불어 LPA에 의한 SKOV-3 세포의 이동도 NADPH oxidase 억제에 의해 저해가 됨을 확인하였다. 이러한 연구결과로 보아 LPA에 의한 활성산소의 형성에는 PI3K/Akt/NADPH oxidase 신호전달체계가 중추적인 역할을 하며 이를 통해 암세포의 이동을 조절한다는 것을 알 수 있었다.

• The Korean Journal of Physiology
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• 제23권2호
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• pp.263-275
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• 1989

Mechanical contractions and electrical activities of the fundic longitudinal and antral circular muscle fibers were investigated in order to elucidate topical differences of gastric motility. K-induced contracture was produced by exposure of muscle strips to high K Tyrode solution. Membrane potential and mechanical contraction were simultaneously recorded by conventional glass microelectrode method and single sucrose-gap technique. All experiments were performed in tris-buffered Tyrode solution which was aerated with $100%\;O_2\;and\;kept\;35^{\circ}C$. The results obtained were as follows: 1) The resting membrane potential of circular muscle cells in the antral region was about 10 mV more negative than that in the fundic region. 2) The membrane potentials decreased almost linearly as the extracellular KCI concentration was increased both in antral circular muscle cells and in fundic longitudinal muscle cells. 3) The thresholdal K concentration of K-contracture was 15 mM (membrane potential, -48 mV) for the antral circular muscle strip and 20 mM for the fundic longitudinal muscle cells. 4) The ratio of membrane permeability coefficient for $Na^+\;and\;K^+,\;P_{Na}/P_K\;({\alpha})$ was 0.065 for antral circular muscle cells and was 0.108 for fundic longitudinal muscle cells. 5) K-contracture of antral and fundic smooth muscle strips showed the contracture composed of phasic and tonic components. The amplitude of the phasic component increased sigmoidally in a dose-dependent manner, whereas that of the tonic component was maximal at a concentration of 40 mM KCI and at the concentrations above or below 40 mM KCI the amplitude was reduced. 6) The inverse relationship between the amplitude of tonic component and extracellular KCI concentration in the range of 40 to 150 mM KCI was more prominent in the antral circular muscle strip than in the fundic longitudinal muscle strip, where the amplitude of the tonic component decreased less steeply and was maintained higher at the same high K concentrations. 7) The tonic component was totally dependent on the external $Ca^{2+}$ and completely abolished by verapamil, while tile phasic component was far less dependent on the external $Ca^{2+}$ and partially suppressed by verapamil. From the above results, the following conclusions could be made. 1) The phasic component of K-contracture is produced both by intracellular $Ca^{2+}$ mobilization and by $Ca^{2+}$-influx from outside, while the tonic component is generated and maintained by the $Ca^{2+}-influx$ through the potential-dependent $Ca^{2+}$ channel. 2) The mechanism of reducing the free $Ca^{2+}$ concentration in the myoplasm seems to be more developed in the antral circular muscle than in the fundic longitudinal muscle. 3) The lower resting membrane potential of the fundic longitudinal muscle cell reflects a relatively high $P_{Na}/P_K$ ratio of about 0.108.

• 대한약리학회지
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• 제30권2호
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• pp.191-203
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• 1994

$K^+$ channel openers (KCOs) are known to have a wide range of effects by opening the $K^+$ channel in plasma membranes of various smooth muscles, cardiac muscle and pancreatic ${\beta}-cell$. In the present study, we investigated the effects of 5 types of KCOs, cromakalim, RP49356, pinacidil, nicorandil and diazoxide on the contractility of isolated rat uterus. All KCOs tested inhibited the uterine contraction induced by 0.2 nM oxytocin in a dose-dependent manner. Individual KCO and its $pD_2$ values were cromakalim 6.5, RP49356 6.3, pinacidil 5.92, nicorandil 4.43 and diazoxide 4.18. The relaxant effects of KCO were inhibited by glibenclamide (0.3, 1 and $10\;{\mu}M$) with $pA_2$ values of cromakalim 6.91, RP49356 6.59, pinacidil 6.55, nicorandil 5.97 and diazoxide 6.37. In addition, the relaxant effect of cromakalim or pinacidil was antagonised by TEA, a non-selective $K^+$ channel blocker, but not by apamin. Contractions induced by low concentration of KCI (< 40 mM) were inhibited by cromakalim $(100{\mu}M)$ and nicorandil $(300{\mu}M)$, but those evoked by higher concentration (> 40 mM) of KCI were little affected. In ovariectomized rat uterus, cromakalim dose-dependently inhibited oxytocin-induced contraction and glibenclamide $(10{\mu}M)$ inhibited the relaxant effect of cromakalim with $pD_2$ and $K_B$ values of 7.48 and $1.26{\times}10^{-7}M$, respectively. In estrogen-primed rat uterus, these values were 6.51 and $1.57{\times}10^{-7}M$, respectively, indicating that the cromakalim is less effective on the estrogen-treated uterine smooth muscle. Our results suggest that the KCO-sensitive $K^+$ channels participate in the motility of uterine smooth muscle and such channels are, at least in part, under the control of estrogen. In addition, our data Indicate that the type of $K^+$ channels activated by KCO is ATP-sensitive $K^+$ channels which is blocked by glibenclamide.

• The Korean Journal of Physiology and Pharmacology
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• 제2권1호
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• pp.85-93
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• 1998

ATP-sensitive $K^+$ channels ($K_{ATP}$) were not identified in gastric smooth muscle cells. However, in tension recording of intact gastric circular muscle, lemakalim of $K_{ATP}$ channels opener in other tissues suppressed mechanical contractions and this effect was blocked by glibenclamide, a specific inhibitor of $K_{ATP}$ channels. The aims of this study were to investigate whether $K_{ATP}$ channels exist in gastric smooth muscle of guinea-pig and to know its physiological role. Whole cell $K^+$ currents activated by lemakalim were recorded from freshly isolated cells with a 0.1 mM ATP, 140 mM KCl pipette solutions. Lemakalim (10 ${\mu}M$) increased inward currents of $-224{\pm}34$ pA (n=13) at -80 mV of holding potential in bath solution contained 90 mM $K^+$. Bath-applied glibenclamide (10 ${\mu}M$) inhibited the lemakalim-activated inward currents by $91{\pm}6%$ (n=5). These lemakalim-activated inward currents were reduced by increased intracellular ATP from 0.1 to 3 mM ($-41{\pm}12$ pA) (n=5). The reversal potential of the glibenclamide- sensitive inward currents was $-5.2{\pm}2.4$ mV (n=3) in external 90 mM $K^+$ and shifted to $-14.8{\pm}3.6$ mV (n=3) in external 60 mM $K^+$, which close to equilibrium potential of $K^+$ ($E_K$). External barium and cesium inhibited the lemakalim-activated inward currents dose-dependently. The half-inhibitory dose ($IC_{50}$) of barium and cesium were 2.3 ${\mu}M$ (n=5) and 0.38 mM (n=4), respectively. 10 mM tetraethylammonium (TEA) also inhibited the lemakalim-activated inward currents by $66{\pm}15%$ (n=5). Both substance P (SP) (5 ${\mu}M$) and acetylcholine (ACh) (5 ${\mu}M$) inhibited lemakalim-activated inward currents. These results suggest that $K_{ATP}$ channels exist in the gastric smooth muscle and its modulation by neurotransmitters may play an important role in regulating gastric motility.

• Asian-Australasian Journal of Animal Sciences
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• 제22권8호
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• pp.1107-1112
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• 2009

The separation of X and Y chromosome-bearing sperm is of use in many aspects of livestock maintenance. In this study, we sought to determine the difference in DNA content between X- and Y-bearing sperm, separate sperm into X- and Y-enriched pools, and assess the efficacy of sorting. Sperm collected from Duroc and miniature pigs were stained with 20.8 $\mu{M}$ Hoechst 33342 and analyzed using a high-speed cell sorter. Measurement of the fluorescence intensity of stained sperm nuclei revealed that the X-bearing sperm of Duroc and miniature pigs respectively contain 2.75% and 2.88% more DNA than Y-bearing sperm. In total, 50.18% of the sperm were assigned to the X-sorted sample and 49.82% was assigned to the Y-sorted sample for Duroc pigs. For miniature pigs, the Xsorted sample represented 50.19% of the population and the Y-sorted represented 49.81% of the population. Duplex PCR was used to evaluate accuracy of sorting. A fast and reliable method for porcine sexing was developed through amplification of the sex-determining region of the Y chromosome gene (SRY). Oligonucleotide primers were designed to amplify the conserved porcine SRY high motility group (HMG) box sequence motif. We found that the primer pair designed in this study was 1.46 times more specific than previously reported primers. Thus, this study shows that the present method can be applied in porcine breeding programs to facilitate manipulation of the sex ratio of offspring and to achieve precise sexing of porcine offspring by amplification of the HMG box of the SRY gene.

• 한국미생물·생명공학회지
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• 제45권2호
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• pp.168-177
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• 2017

Strain A28-5는대한민국 제주도 연안의 해수샘플로부터 고체배지내 xylan과 agar를 분해하는 균주로 분리되었다. Strain A28-5는그람 음성균으로 한 개의 polar flagella로 운동성을 갖는 $Na^+$ 이온 요구성 균주로 분석되었다. 또한 ampilcillin과 thiostreptone 등의 항생제에 내성을 보였다. Genome 내 G+C content는 43.96%이고, Menaquinone-7 (MK-7)을 predominant quinone으로 함유하고 있었다. Strain A28-5의 세포벽을 구성하는 주요 지방산은 $C_{16:1}$ ${\omega}7c/iso-C_{15:0}$ 2-OH (23.32%), $C_{16:0}$ (21.83%), $C_{18:1}$ ${\omega}7c$ (17.98%)였다. strain A28-5의 16S rRNA gene sequence는 Catenovulum agarivorans YM01와 가장 높은 상동성(98.94%)을 보였으며, Neighbor-Joining phylogenetic tree 제작을 통해서 Catenovulum agarivorans YM01와 가장 높은 근연관계를 보이는 것을 증명하였다. Catenovulum agarivorans YM01과의 DNA-DNA hybridization 분석을 통하여 A28-5을 Catenovulum 속의 신종으로 분류하여Catenovulum jejuensis A28-5로 명명하였다. 이 균주의 액체배양으로부터 준비된 두 종류의 조효소를 이용한 xylan 또는 agarose와의 효소반응액을 Thin layer chromatography로 분석하여 각각 tetramer와 hexamer의 xylooligosaccharides와 (neo)agarooligosacchardes가 생산되는 것을 확인하였다.

• 사상체질의학회지
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• 제12권1호
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• pp.186-200
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• 2000

소양인(少陽人) 인동등지골피탕(忍冬藤地骨皮湯)이 중소(中消) 합병증(合倂症)에 미치는 영향(影響)을 살펴보고자 하였고 결과(結果)는 다음과 같다. 1. 장관운동(腸管運動)을 억제(抑制)하였다. 2. Acetic acid에 의한 writhing syndrome을 억제(抑制)하였다. 3. Evans blue에 의한 모세혈관투과성(毛細血管透過性)을 억제(抑制)하였다. 4. Histamine에 의한 급성족부종(急性足浮腫)을 억제(抑制)하였다. 5. Cotton pellet에 의한 육아종(肉芽腫) 형성(形成)을 억제(抑制)하였다. 6. 흉선세포(胸線細胞) 및 비장세포(脾臟細胞)의 세포생존율(細胞生存率)을 증가(增加)시켰다. 7. 흉선세포(胸線細胞)로부터 ${\gamma}-IFN$ 및 IL-2의 분비(分泌)를 억제(抑制)하였다. 8. 비장세포(脾臟細胞)로부터 IL-4의 분비(分泌)는 증가(增加)시켰으나, $TNF-{\alpha}$ 및 $IL-1{\beta}$의 분비(分泌)는 억제(抑制)하였다. 9. 복강(腹腔) 대식세포(大食細胞)로부터 $TNF-{\alpha}$, $IL-1{\beta}$ 및 nitric oxide의 분비(分泌)를 억제(抑制)하였다. 10. 복강(腹腔) 대식세포(大食細胞)의 lucigenin chemiluminescence 및 FITC-conjugated E. cole engulfment를 촉진(促進)하였다. 이상의 실험(實驗) 결과(結果) IJTE는 장관운동(腸管運動)을 억제(抑制)하였으며, 진통(鎭痛) 및 항염작용(抗炎作用)을 나타냈고, 이들 작용은 면역세포(免疫細胞)로부터 $TNF-{\alpha}$, $IL-1{\beta}$ 및 nitric oxide의 생성(生成)을 억제(抑制)하기 때문인 것으로 추정된다. 따라서 이러한 기전(機轉)에 의하여 인동등지골피탕(忍冬藤地骨皮湯)은 중소(中消) 합병증(合倂症)의 하나인 염증(炎症)을 억제(抑制)하는데 유효(有效)한 것으로 사료(思料)된다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권21호
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• pp.9327-9333
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• 2014

Breast cancer is the second most common cancer and second leading cause of cancer deaths in women. Phosphatidylinositol-3-kinase (PI3K)/AKT pathway mutations are associated with cancer and phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene mutations have been observed in 25-45% of breast cancer samples. Insulin growth factor binding protein-5 (IGFBP-5) can show different effects on apoptosis, cell motility and survival in breast cancer. We here aimed to determine the association between PIK3CA gene mutations and IGFBP-5 expressions for the first time in breast cancer patients. Frozen tumor samples from 101 Turkish breast cancer patients were analyzed with high resolution melting (HRM) for PIK3CA mutations (exon 9 and exon 20) and 37 HRM positive tumor samples were analyzed by DNA sequencing, mutations being found in 31. PIK3CA exon 9 mutations (Q546R, E542Q, E545K, E542K and E545D) were found in 10 tumor samples, exon 20 mutations (H1047L, H1047R, T1025T and G1049R) in 21, where only 1 tumor sample had two exon 20 mutations (T1025T and H1047R). Moreover, we detected one sample with both exon 9 (E542Q) and exon 20 (H1047R) mutations. 35% of the tumor samples with high IGFBP-5 mRNA expression and 29.4% of the tumor samples with low IGFBP-5 mRNA expression had PIK3CA mutations (p=0.9924). This is the first study of PIK3CA mutation screening results in Turkish breast cancer population using HRM analysis. This approach appears to be a very effective and reliable screening method for the PIK3CA exon 9 and 20 mutation detection. Further analysis with a greater number of samples is needed to clarify association between PIK3CA gene mutations and IGFBP-5 mRNA expression, and also clinical outcome in breast cancer patients.

• 생명과학회지
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• 제27권6호
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• pp.673-679
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• 2017

미세소관을 따라 이동하는 모터단백질들은 세포내 물질수송에 필수적인 역할을 한다. Kinesin 1은 세포내에서 미세소관을 따라 움직이는 모터단백질로서 다양한 소포, mRNA, 그리고 단백질의 세포내 수송에 관여한다. Kinesin 1은 2개의 장쇄단위체(KHCs, 또는 KIF5s)와 2개의 경쇄단위체(KLCs)로 구성되어 있다. KIF5s는 N-말단에 모터도메인을 가지고 있고 C-말단의 운반체 결합도메인을 통해 다양한 운반체와 결합한다. 본 연구에서 KIF5B와 결합하는 단백질을 분리하기 위하여 효모 two-hybrid 탐색을 수행한 결과 미세소관의 plus end 결합단백질인 cytoplasmic linker protein 170 (CLIP-170)을 분리하였다. CLIP-170의 coiled-coil 도메인은 KIF5B의 운반체 결합도메인과 결합하였다. 또한 CLIP-170은 KIF5A와 KIF5C와도 결합하였다. 그리고 glutathione S-transferase (GST) pull-down을 통해 KIF5s와 CLIP-170이 단백질수준에서 결합함을 확인하였다. 생쥐 뇌파쇄액을 KIF5B 항체로 면역침강한 결과 CLIP-170이 같이 침강함을 확인하였다. 이러한 결과들은 kinesin 1이 세포내에서 CLIP-170을 운반함을 시사한다.