• BMB Reports
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• 제28권2호
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• pp.184-190
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• 1995

Functional study of the gap junction channel has been hindered by its inaccessibility in situ. Identification of forms of this channel in artificial membrane has been elusive because of the lack of identifying channel physiology. Connexin32 forms gap junction channels between neighboring cells in rat liver. Connexin32 was affinity-purified using a monoclonal antibody and reconstituted into artificial phospholipid vesicles. The reconstituted connexin32 formed channels through the vesicle membrane that were permeable to sucrose (Stokes radius: $5{\AA}$). The permeability to sucrose was reversibly reduced by acidic pH. In addition, the pH effect on the permeability to sucrose fit well with by the Hill's equation (where, n=2.7 and pK=6.7).

• 생명과학회지
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• 제18권6호
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• pp.764-769
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• 2008

커넥신(connexin) 32 유전자의 돌연변이가 씨엠티엑스(CMTX, X-linked Charcot-Marie-Tooth) 질환과 관련이 있다. 현재까지 300여개 이상의 돌연변이가 보고가 되었으나 이 질환에 대한 상세한 분자병리학적 원인을 거의 알려져 있지 않고 있다. 여러 연구를 통해서 커넥신 세포막채널이 간극결합채널이 갖고 있는 대부분의 생물리학적 특성을 갖고 있는 것으로 판명되었다. 이번 연구에서는 씨엠티엑스 질환과 관련된 두 개의 돌연변이체를 선정하여 간극결합채녈 대신 돌연변이체로 구성된 커넥신 세포막채널을 이용하여 단일채널수준에서 이들 돌연변이체의 특성을 조사하였다. M34T 돌연변이 세포막채널의 생물리학적 특성은 이들로 구성된 돌연변이 간극결합채널의 특성과 거의 유사하였다. 더욱이, 돌연변이 세포막채널을 이용한 연구를 통해서 간극결합채널을 이용한 연구에서는 밝혀지지 않았던 개폐극성의 역전, 빠른 개폐의 소실과 느린 개폐의 생성과 같은 새로운 사실을 알게 되었다. T86C 돌연변이 세포막채널 또한 이의 모체가 되는 커넥신 32 세포막채널과 유사한 특성을 갖고 있음을 알게 되었다. 이상의 결과를 통해서 커넥신 세포막을 이용한 연구가 씨엠티엑스 질환의 돌연변이체를 연구하는데 매우 유용할 것으로 생각된다.

• 대한수의학회지
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• 제45권4호
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• pp.517-525
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• 2005

This study was performed to evaluate using wild-type (WT) and $C{\times}32$ knockout (KO) mice if lack of cell to cell communication by connexin 32 gap junction enhances the benzene-induced hematotoxicity and hemopoietic tumor development. The WT and $C{\times}32$ KO mice were exposed to 300 ppm of benzene for 6 hours/day, 5 days/week for a total of 26 weeks by inhalation, and then sacrificed to evaluate the toxicities of hemopoietic organs or allowed to live out their life span to evaluate the hemopoietic tumor incidence. The significant increase and decrease of organ weight were respectively noted in spleen and thymus of both WT and $C{\times}32$ KO mice without significant difference between the genotypes. Histopathologically, benzene exposure for 26 weeks induced the morphological changes in hemopoietic organs, characterized by fat cell accumulation in the bone marrow and extramedullary hemopoiesis in the spleen. The fat cell accumulation was, compared with that of WT mice, considerably exacerbated in the $C{\times}32$ KO mice. However, no significant difference was observed in the changes of hematological values and bone marrow cellularity as well as in the onset and incidence of hemopoietic tumors between WT and $C{\times}32$ KO mice. In conclusion, this study indicated little significant role of the cellular communication by $C{\times}32$ gap junction in the action mechanism of benzene hematotoxicity and leukemogenicity.

• 한국수산과학회지
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• 제33권6호
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• pp.565-571
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• 2000

기존의 Cx 배열을 참고로 종내${cdot}$종간을 통하여 잘 보존되어져 있는 영역에서 primer를 설계하고, 은어의 난소를 재료로 하여 PCR을 실시하였다. 증폭된 cDNA 단편을 이용하여, 5'RACE 및 3'RACE법에 의해 미지의 영역을 cloning하여 난소에서 발현하는 Cx cDNA의 전염기배열을 결정하였다. 기존의 Cx 배열과 상동성을 비교한 결과, 대서양산 민어의 Cx32.7과 $63.8{\%}$, bovine의 Cx44와 $61.6{\%}$ 및 대서양산 민어의 Cx32.2와는 $56.7{\%}$의 상동성이 나타났다. 본 cDNA는 35,028 Da의 분자량을 code하는 open reading frame (ORF)으로 구성되어 있어, 은어 Cx35로 명명되었다. 또한 아미노산 배열의 친수성${\cdot}$소수성 영역의 분포예측 결과, 4곳의 소수성 영역과 4곳의 친수성 영역을 교차하는 전형적인 Cx의 구조와 일치하였으며, Cx family의 공통${\cdot}$필수적인 배열인 제1세포외 domain의 consensus 배열 및 제2세포외 domain의 consensus 배열도 존재하였다.

• 생명과학회지
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• 제17권11호
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• pp.1517-1522
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• 2007

간극결합(intercellular channel)은 인접하는 두 세포사이에 형성된 이온채널이며 이를 통해서 각종 이온, 이차 신호전달물질, 그리고 1 kDa 미만의 대사물질들이 통과한다. 아울러, sodium 혹은 potassium 이온채널처럼 반쪽의 간극결합(connexon 혹은 hemichannel)도 세포막채널로서 작용을 한다. 현재까지 간극결합을 구성하는 connexin (Cx) 단위체는 26종류 이상이 확인되었다. 이 가운데, Cx32, Cx38, Cx46 그리고 Cx50 만이 간극결합채널뿐만 아니라 세포막채널로서도 기능을 수행한다. Xenopus oocytes에서 connexin 38 (Cx38)이 발현하는 것으로 알려져 있지만 Cx38의 생물리학적 특성이 단일채널수준에서 연구가 진행된 경우는 없다. 이번 연구에서는 Cx38 채널의 생물리학적 특성, 즉 전압-의존적 개폐와 투과성(전기전도도와 이온선택성)을 알아보고자 단일채널기록을 수행하였다. Cx38 hemichannel은 전압-의존적인 빠른 개폐와 느린 개폐의 특성을 보였다. 양성전압 환경에서는 Cx38 채널이 낮은 열릴 확률(open probability)로 빠른 개폐가 유도된 반면, 음성전압에서는 느린 개폐가 높은 열릴 확률로 유도되었다. bi-ionic 실험을 통하여, Cx38 채널은 양이온보다 음이온을 더 선택 적으로 통과시킨다는 점을 알게 되었다. Cx38의 아미노산서열을 살펴보면, 아미노말단부위에 전하를 띠는 5개의 아미노산 잔기가 존재한다. 앞으로 이들 잔기를 치환시킨 돌연변이 Cx38 채널을 이용하여 과연 이들 아미노산 부위가 전압-의존적 개폐와 투과성에 관여하는 지 여부를 조사하는 연구는 매우 흥미로운 결과를 도출할 것으로 기대한다.

• Journal of Animal Science and Technology
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• 제54권6호
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• pp.419-425
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• 2012

The coordinated action of the detrusor muscle cells in the urinary bladder is governed by cell-cell communication through gap junction, consisted of connexin (Cx) molecules. Even though a number of researches have been mostly focused on expressional changes of a few Cx isoforms in clinically dysfunctional condition of the bladder, less attention has been paid for investigation of Cx isoforms present in the bladder. Using real-time PCR analysis, the present study examined Cx isoforms expressing in the male rat bladder during postnatal period. Also, expressional patterns of Cx isoforms were evaluated in the bladder at different postnatal ages. Of a total of 13 Cx isoforms tested in the present study, we were able to detect mRNAs of 6 Cx isoforms in the rat urinary bladder, including Cxs 31, 31.1, 32, 37, 40, and 45. The transcript levels of Cxs 31, 31.1, 37, 40, and 45 were gradually increased from 1 week of age until 25 days of age, followed by transient decreases at 45 days of age. However, abundance of Cx32 transcript was drastically increased at 15 days of age, followed by a sharp drop at 45 days of age. These results indicate that differential expression of Cx isoforms in the bladder during postnatal development would be necessary for maintaining proper function of the bladder. A question remains to be answered if significant decreases of transcript levels of some Cx isoforms at the elderly are associated with age-dependent dysfunction of the bladder.

• Archives of Pharmacal Research
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• 제19권4호
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• pp.264-268
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• 1996

Panax-ginseng saponin has been known to exert various pharmacological effects on cellular metabolism. This study was performed to determine the effect of ginseng saponin on gap junction channel-mediated intercellular communication, using an established in vitro system of reconstituted gap junction channels. Gap junction channels are a specialized plasma membrane fraction, which are permeable to relatively large water-soluble molecules. The sucrose permeable property of reconstituted gap junction channels was completely inhibited with 0.1 % (w/v) of ginseng saponin. We also compared the effect of ginseng saponin with that of Triton X-100, a nonionic detergent, on the same system. Triton X-100 showed significantly different effect on sucrose-permeability of gap junction channel from that was affected by ginseng saponin. The structures of liposomes containing gap junction channels was significantly destroyed by Triton X-100.

• 한국발생생물학회지:발생과생식
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• 제20권3호
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• pp.237-245
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• 2016

Direct communication between neighboring cells via gap junction in tissue is important for maintenance and regulation of its physiological functions. Each epididymal region has different composition of cell types. It is well recognized that the epididymis is a steroid hormone-responsive tissue. The present study was designed to determine the effect of estradiol benzoate (EB) or flutamide exposured at the early postnatal age on the expression of connexin (Cx) isoforms in the caudal epididymis. The EB or flutamide was subcutaneously administrated to male Spragure Dawley rat at 7 days of age, and expressional changes of Cx isoforms in the adult corpus epididymis were determined by quantitative real-time PCR. The treatment of low-dose EB resulted in decreases of Cx30.3, Cx31.1, Cx37, and Cx45 expression but caused an increase of Cx32 expression. Exposure to high-dose EB led into expressional increases of Cx31, Cx31.1, Cx32, Cx40, and Cx43, even though a decrease of Cx37 expression was found with a high-dose EB treatment. A low-dose flutamide induced increases of Cx31, Cx31.1, Cx32, and Cx43 expression but a decrease of Cx37 expression. Expression of most Cx genes were significantly increased by a high-dose flutamide, while no expressional change of Cx26 and Cx40 was detected by a high-dose flutamide. These results indicate that expression of Cx isoforms in the caudal epididymis is altered by exposure to steroidal compounds at the prepubertal age. It is suggested that a contact with environmental exogenous materials during the early postnatal period would lead to alteration of epididymal functions at the adult.

• 대한수의학회지
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• 제43권4호
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• pp.649-656
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• 2003

We have a cultured method to grow rat mammary epithelial cells (RMEC) for 1 to 14 days in 1:1 mixture of Dulbecco's Modified Eagle Medium: Nutrient and F-12 (DMEM/F-12) containing 10% fetal bovine serum (FBS), human EGF, insulin, hydrocortisone, human transferrin and $17{\beta}$-estradiol in vitro. We were able to isolate and distinguish two cell types, luminal epithelial cells and myoepithelial cells, from primary clutures of RMEC. Immunocytochemical stains were used to distingusih luminal epithelial cells and myoepithelial cells. Peanut lectin (PNA) was stained in most alveolar epithelail cells and luminal epithelial cells of rats, while Thy-1.1, a maker of potential rat mammary myoepithelial cells, was expressed in myoepithelial cells in the rat. Also, we examined the expression patterns of three types of gap junction proteins, connexin 26 ($C{\times}26$), connexins 32 ($C{\times}32$) and connexin 43 ($C{\times}43$) by immunocytochemistry and western blot analysis. In the cell types, the results show that at the early stage of culture, luminal epithelial cells were increased and these cells were surrounded by myoepithelial cells. At the late stage of culture, luminal epithelial cells were decreased, in contrast myoepithelial cells were increased. In the expression pattern of gap junction, $C{\times}26$ maintained it's expression until day 3, but afterwards gradually decreased in intensity. Expression of $C{\times}32$ remained until day 5, then decreased slightly. $C{\times}43$ gradually increased untill the middle time of culture then decreased in intensity. These results suggest that connexins may be important for the control of growth in rat mammary epithelial cell types.

• 한국발생생물학회지:발생과생식
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• 제19권4호
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• pp.217-226
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• 2015

A proper development of the epididymis during the early postnatal development is required for successful fertility in the adult male. Direct cell-cell communication via connexin (Cx) molecules is a common way of cellular interactions to achieve normal development of a given tissue consisting of different cell types. The present research was attempted to determine the effect of exogenous exposure to estrogenic agonist or antiandrogen at the weaning age on expression of Cx isoforms in the adult corpus epididymis. Male rats were subcutaneously administrated with estradiol benzoate (EB) or flutamide (Flu) at the weaning age. The tissue was collected at 4 months of age. Expressional levels of Cx isoforms were determined by a quantitative real-time PCR. Statistical comparison showed significant increases of Cxs31, 32, 37, 40, and 43 transcript amounts by a treatment of $0.015{\mu}g$ of EB /kg body weight (BW). A treatment of $1.5{\mu}g$ of EB /kg BW caused a significant decrease of Cx43 gene expression but increases of Cxs26, 31, 32, 37, and 40 transcript levels. Exposure to $500{\mu}g$ of Flu/kg BW induced an increase of Cx37 expression but significant decreases of Cxs43 and 45 mRNA levels. Expression of Cx37 was increased by a treatment of 5 mg of Flu/kg BW, while transcript levels of Cxs26, 30.3, 31, 31.1, 32, and 43 were significantly decreased by same treatment. These results demonstrate that exposure to steroidal compounds at the early developmental age alters expression of Cx isoforms in the adult corpus epididymis.