닭 조직에 따른 Nebulin Isoform 단백질의 특이적 발현

김영희,김정락 대한의생명과학회 2000 Journal of biomedical laboratory sciences Vol.6 No.3

대부분 척추동물에서 골격근 내 filament들의 길이 조절은 근 수축 기작의 구조를 이해하는데 중요한 단서가 된다. Nebulin은 thin filament의 전체에 걸쳐있는 거대한 단백질로 골격근에만 특이적으로 존재하는 것으로 알려져 왔다. 본 연구에서는 닭의 근육과 비근육 조직에서 nebulin isoform 단백질들을 확인하기 위하여 전기영동과 immunoblot의 방법을 이용하였다. 각 조직의 단백질들은 soluble과 insoluble fraction으로 분리 준비하였다. 실험결과, 닭의 근육과 비근육 조직들에서 조직 특이성을 나타내는 다양한 nebulin isofrm 단백질들이 확인되었다. Nebulin은 성계의 골격근에서 500 kDa 정도의 크기로 나타났고, nebulett은 계배와 성계의 심장근에서 107 kDa 정도로 발현되었다. 그리고 계배의 비근육 조직인 뇌에서 380 kDa 정도의 거대 단백질이 확인되었다. 이 단백질은 뇌 조직의 soluble fraction에서 인지되었다. Nebulin isoform 단백질들이 서로 다른 조직에서 발현되는 양상을 보아 서로 다른 독자적인 기능을 가질 것으로 추정된다. The lengths of thick and thin filaments in the sarcomeres of most vertebrate skeletal muscles are precisely regulated and are important structural parameters in understanding muscle contraction. Nebulin is a usually large protein that spans the whole length of thin filaments in the sarcomeres of skeletal muscles. In this paper we used SDS-PAGE and immunoblot to identify nebulin isoform proteins in muscle and non-muscle tissues. We prepared embryonic chicken tissues including skeletal muscle, cardiac muscle, smooth muscle, brain liver to compare nebulin isoform proteins. The proteins were divided into soluble and insoluble fraction. As a result, we identified tissue specific expression of various nebulin isoform proteins in muscle and non-muscle tissues of chicken. Nebulin was detected in skeletal muscle of adult chicken about 500 kDa. Nebulett was expressed in cardiac muscle of embryonic and adult chicken about 107 kDa. A giant protein with molecular mass of about 380 kDa was identified in brain of non-muscle of chicken. This giant protein was detected in the soluble fraction of chicken embryo. The unequal distribution of the nebulin isoform proteins suggests tissue specific regulation of the isoform expression and indicates a functional specialization of the encoded isoform subtypes.

계배 근세포의 분화단계에 따른 cDNA library 제작과 분석

김상해,이채관,박수정,정선미,김정락,강성구 인제대학교 1991 仁濟論叢 Vol.7 No.2

계배근세포의 분화단계에서 발현되는 mRNA들에 대한 cDAN library들을 제작하였다. 분화전과 후의 cDNA들을 서로 상쇄시켜 분화과정의 초기단계에 특이적인 한종의 cDNA를 분리하였다. 이 cDNA의 크기는 약 0.8kb이고 하나의 HindIII 제한효소 자리를 가지며 EcoR I, BamH I, Xba I, Kpn I, Sma I, 그리고 Pst I 자리는 포함하지 않았다 The cDNA libraries for mRNAs expressed on differentiation stages in chick embryonic muscle cells were constructed. A species of cDNA specific on early stage of differentiation process was isolated by subtracting ones after differentiation from cDNAs before differentiation of muscle cells. Size of the cDNA is about 0.8kb and it contains one HindIII site but no EcoR1, BamHI, XbaI, KpnI, SmaI, and PstI site.

배양 근 세포의 분화 Tunicamycin과 o-Phenanthroline의 효과

김정락 인제대학교 1987 仁濟論叢 Vol.3 No.2

The effect of tunicamycin(50ng/ml) and o-phenanthrolin(5㎍/ml) as fusion inhibitor on the myoblast in culture was investigated. Confluent myoblast exposed to tunicamycin, an inhibitor of dolichol-phosphate-mediated glycosylation of proteins at asparagine-linked site, or to o-phenanthrolin, a chelator of divalent metal cations, did not fuse or express creatine phosphokinase, acetylcholin receptor, myosin heavy chain, or α -actin. o-Phenanthrolin did not perturb myoblast adhesion but rather blocked the fusion of aggregated cells. By contrast, tunicamycin blocked elongation step.

펄프지료의 pH가 고해에 미치는 효과

김창락,문상환,김재옥,김철환,박종열 경상대학교 농업생명과학연구원 2003 농업생명과학연구 Vol.37 No.4

펄프 섬유를 이용해서 종이를 제조하기 위해서는 반드시 섬유를 물에 현탁시키는 해리와 섬유 벽에 물리적 변화를 부여하는 고해의 과정을 거쳐야 하고, 이 과정에서 섬유는 팽윤 과정을 겪게 된다. 섬유가 물에 의해 팽윤이 잘 될수록 유연성이 좋아지기 때문에 종이의 물리적 성질이 훨씬 향상된다. 펄프를 물에 현탁시키는 전처리 과정에서 NaOH를 첨가하여 침지과정에서 섬유의 팽윤이 더 잘되게 하기 위함인데, NaOH의 처리량을 증가시킴에 따라 고행 동안 섬유벽의 팽윤이 촉진되어 종이의 강도적 성질이 향상되는 것을 확인할 수 있었다. Pulp fibers must be disintegrated and refined before papermaking. During refining, internal fibrillation of fiber walls are generated, leading to fiber wall swelling. Owing to improved fiber flexibility, strength properties of paper are remarkably improved. This study aimed to explore how NaOH addition to pulp suspension before refining would affect fiber swelling in addition to paper properties. As the addition ratio of NaOH increased, strength properties of paper were greatly improved due to the increase of fiber wall swelling. The extent of fiber wall swelling according to pH was observed by paraffin embedding and physical sectioning. In conclusion, it became evident that higher pH in fiber suspension gave positive contribution to fiber wall swelling during refining.

계배 배양 근원 세포 항원에 대한 단일 클론 항체의 생산

박수정,강동수,김정락 인제대학교 1990 仁濟論叢 Vol.6 No.1

Monoclonal antibodies raised in mouse BALB/c against cultured chick myoblast were found to exhibit reactivity toward myoblast proteins of 109 kDa, 19 kDa, 18 kDa and 15 kDa and others. Especially the levels of 109 kDa, 18 kDa and 16 kDa proteins decreased during the course of myogenesis in culture and between embryo and adult muscle. These antibodies delayed the differentiation and enhanced the cell division of myoblast when added to culture media. Their effects were saturated by adding one fiftieth of media and accompanied with the increments of 200 kDa, 29 kDa and 15 kDa myoblast proteins, the decrement of 25 kDa protein, and the new synthesis of 57 kDa protein.

근원세포 분화조절 유전자의 클로닝

강성구,이채관,김상해,김정락 인제대학교 1992 仁濟論叢 Vol.8 No.2

배아의 근원세포는 분화가 진행 됨에 따라 성체의 골격근 세포로 된다. 이러한 분화단계에는 특정 유전자가 관여할 것으로 추정되며 이전 실험에서 0.8Kb의 cDNA를 확인하였다 (Kang et al., 1992). 이러한 특이 유전자는 계배 부화 9일째부터 발현이 되었으며 성체에서는 발현이 되지 않았다. 특히 12일과 14일 계배의 뇌,간,신장조직에서는 유전자 발현이 되지 알았으며 단지 가슴근육조직에서만 유전자 발현이 되었다. 이 유전자는 계배 골격근 세포의 분화에서 중요한 역할을 할 것으로 추정된다. Embryonic myoblasts become determined to differentiate into distinctive types of muscle cells. Skeletal muscle cell is one of them. We studied the expression of muscle specific 0.8Kb cDNA related to the differentiation of chick embryonic myoblast in various stage of development and in organs. The gene was expressed from 9 day old chick embryo stage, but was not expressed at adult breast muscle. Also the gene was not expressed in brain, liber, kidney but expressed in 12 and 14 day old chick embryonic breast muscle. Therefore, this muscle specific gene(0.8Kb) may have an important role in regulating the diffrentiation of chick embryonic myoblast.

근원세포의 융합과 연관된 표면 단백질의 확인

박수정,주영미,김정락 인제대학교 1991 仁濟論叢 Vol.7 No.2

배양 근원세포 분화에 따른 세포융합에 직접적으로 관여하는 단백질을 찾기 위해 48 시간 배양한 근원세포를 항원으로 BALB/C mouse에 면역시켜 얻은 spleen cell과 myeloma cell(P3U-1)을 융합시켜 근원세포와 반응하는 항체를 분비하는 hybridoma를 얻었다. 세 차례 한계 희석 후 계대배양하여 분리한 clone들이 분비하는 항체를 근원세포 배양액에 첨가하여 근원세포의 융합을 억제하는 효과가 있는 단일클론항체인 MAb MII-3J3l을 얻었다. MAb MII-3J3l의 효과는 2 ㎍/ml의 농도에서 세포분열에 영향이 없이 근원세포의 융합을 완전히 억제하였으며 이 효과는 적어도 48 시간 지속되었고 항체를 제거할 경우 융합이 재개되어 가역적인 억제효과를 나타내었다. MAb MII-3J31이 인지하는 단백질은 38 kDa로 근원세포 표면을 trypsin 처리하였을 때 잘리워 나감으로 표면단백질로 추정된다. The present study describes the production of monoclonal antibodies against cultured chick myoblast to pursue a protein may play a critical function in muscle cell fusion. Among a panel of monoclonal antibodies, MAb MII-3J3l inhibits myoblast fusion completely, while cell growth proceeds normally and its effect is reversible. The antigen reactive with MAb MII-3J31 isolated by radioimmunoprecipitation and detected by immunoblot was found a 38 kDa protein. For trypsin-treated cell lost its antigenic determinant, the protein may be located in the cell surface.

C-Terminal Region of Ankyrin-B Interact with Z-Line Portion of Titin

Kim, Myong-Shin,Kim, Hyun-Suk,Park, Eun-Ran,Lee, Yeong-Mi,Lee, Min-A,Kim, Ji-Hee,Choi, Jae-Kyong,Ahn, Seung-Ju,Min, Byung-In,Shon, Myeong-Hwan,Choi, Jang-Seok,Kim, Chong-Rak The Korean Society for Biomedical Laboratory Scien 2006 Journal of biomedical laboratory sciences Vol.12 No.4

Ankyrins are a ubiquitously expressed family of intracellular adaptor proteins involved in targeting diverse proteins to specialized membrane domains in both the plasma membrane and the endoplasmic reticulum. We described here that the C-terminal domain of ankyrin-B interact specifically with Z-line portion of titin in yeast two-hybrid analysis, in vitro pull-down assays and localization experiments in COS-7 cells. In this study we provide the first experimental evidence that Z-line portion of titin is necessary for the localization of ankyrin-B and ankyrin-B links between the sarcolemma and the myofibril in costameres.

Inhibition of Myoblast Differentiation by Polyamine Depletion with Methylglyoxal Bis ( guanylhydrazone )

Kim, Han Do,Kang, Ho Sung,Cho, Hwa Jeong,Kim, Byeong Gee,Kim, Chong Rak 생화학분자생물학회 1977 BMB Reports Vol.28 No.3

The role of polyamines in skeletal myoblast differentiation was investigated using the polyamine metabolic inhibitor methylglyoxal bis(guanylhydrazone)(MGBG). Concentrations of intracellular free spemnidine and spemline increased 2 to 2.5-fold at the onset of myoblast fusion. The synthesis of actin, and creative kinase activity both dramatically increased during myotube formation. However, MGBG at a concentration of 0.5 mM not only abolished the increase of intracellular free polyamines, but also reduced cell fusion to almost half the level of untreated cells, without noticeable morphological alteration. The production of actin, and creative kinase activity were almost completely abolished by MGBG. The inhibition of myoblast fusion by MGBG was partially recovered with 0.1 mM of spermidine or spermine added externally. Results indicate that polyamines are necessary for normal myoblast differentiation. Since the first indication of myoblast differentiation is alignment of muscle cells and membrane fusion of adjacent cells, and since polyamine depletion completely inhibited the synthesis of acttn, which might be associated with membranes, polyamine might be involved in myoblast differentiation through membrane reorganization events.

Alteration of Ca^2+ release channel functions in sarcoplasmic reticulum of pressure-overload induced hypertropic rat heart

Kim, Chong-Rak,Kim, Do Han 인제대학교 1993 仁濟論叢 Vol.9 No.2

This study tested the hypothesis that alteration inCa2+ release functions occur with the development of global, left ventricular (LV) hypertrophy induced by pressure-overload. The left ventricular hypertrophied rat hearts were induced by abdominal aorta constriction and the extent of hypertrophy was verified by measuring LV weight (wt)/body wt, LV wt/tibial length and by examining isoforms of myosin heavy chain. The SR vesicles of sham-operated control and hypertrophied rat heart LV were used to study Ca2+ loading (x± SE, ㎎/g wet muscle) of sham and mildly hypertrophied LV(22% hypertrophy) were similar (2.48±0.60 vs 2.65±0.14), whereas active Ca2+ fluxes, while the whole muscle homogenates were used for ryanodine binding. The SR protein yields (x ± SE, nomol/㎎) measured at 0.2μM Ca2+ was significantly lower in the hypertrophied LV than in sham LV(8.6 ± 0.6, n=15 vs. 11.6 ± 1.0, n= 19, p<0.05), Severely hypertrophied hearts(76% hypertrophy) showed significantly lower Ca2+ - activated ryanodine binding (Vmax : 57.3 ± 8.9, n=5 vs. 89.3±7.9 pmol/g, n=4, p<0.03) without significat change in Ca2+ affinity(Km : 0.055±0.005, n=5 vs. 0.055±0.03 μM, n=4). Studies of doxorubicin- or caffeine-induced Ca2+ release (x ±SE) at 5 μM doxorubicin or 0.5mM caffeine was higher in mildly hypertrophied LV SR than in sham LV SR (doxorubicin : 11.4 ± 2.0, n=3 vs. 3.6± 1.8, n=6 ; caffeine ; 10.3 ± 1.8, n=4 vs. 3.6 ± 1.9, n=4). Similarly, % increase in ryanodine binding by 5μM doxorubicin or 0.5 mM caffeine was higher in hypertrophied LV homogenates than in sham. Our results suggest that pressure-overload induced left ventricular hypertrophy is associated with both quantitative and qualitative changes in SR Ca2+ release functions.