• The Korean Journal of Zoology
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• v.38 no.4
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• pp.483-489
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• 1995

Retinoic acid was found to block membrane fusion of chick embryonic myoblasts in culture. This effed was dosedependent and could he reversed upon removal of the agent from the culture medium. Furthermore, the retinoic acid-mediated inhibition of membrane fusion was observed with the fusion competent cells but not with the cells that had already been committed for fusion, indicating that the effect of RA is differentiation stage-specific. However, retinoic acid showed little or no effect on the ability of the cells to form bipolar shape and to align along their axes. Neither the cell proliferation nor accumulation of muscle specific proteins, such as creatine kinase and tropomyosin, was impaired significantly. On the other hand, retinoic acid blocked the differentiation time~ependent loss of fibronectin, whose process is prerequisite for myoblast fusion. These results suggest that retinoic add acts as a specific inhibitor of membrane fusion by preventing the loss of fibronectin from the differentiating myoblasts.

• The Korean Journal of Zoology
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• v.34 no.1
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• pp.31-43
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• 1991

In the present study, the localization of myosin and the pathway of myofibrilogenesis of skeletal muscle cells in culture were investigated by immunocytochemical methods including immunoelectron microscopy and hybridoma formation. There were manly free ribosomes in the cytosol of the myoblast. At 48 hr of the culture, the myofilaments started to form and at 72 hr began to assemble to form myoabrils. At this time the Z band appeared, but the sarcomeres did not link together. Aker 96 hr of culture, adjacent sarcomeres linked together and shotved the typical striation of the skeletal muscle fiber. Myosin was synthesized in free ribosomes at 24 hr of culture and localized at many myofilaments at 48 hr and assembled mvoabrils at 72 hr and at 120 hr of the culture, myosin was localized at thick filaments of the A band.

• The Korean Journal of Zoology
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• v.26 no.4
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• pp.235-251
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• 1983

Several approaches have been made to access the mechanism of fusion in chick myoblast in vitro. Lactoperoxidase-catalyzed iodination was applied to labell cell surface proteins during myogenesis. Quantitative as well as qualitative changes were observed in $^131$I surface components of prefusion and postfusion cells. Two proteins with a molecular weight of 165K and 93K daltons were observed to appear at the onset of fusion as compared to prefusion stage. At the same time, 245K dalton protein decreased whereas the low molecular weight proteins increased consistently. The decrease of high molecular weight proteins appears to be associated with the cell cycle of myoblast during differentiation. The increased appearance of low molecular weight proteins might be due to the proteolytic cleavage of the high molecular weight proteins. Examination of intracellulr cAMP levels during fusion has revealed that a large but transient increase in cAMP occurs before the onset of fusion. This result suggests a causal relationship between the increase of cAMP and the onset of fusion, and further, that differentiating myoblasts are synthronized to a high degree. During the course of myoblast differentiation, at least four lowe molecular weight proteins, which different from major surface proteins iodinated, were identifiable in the culture medium. These proteins could be ascribed to be released from the membrane by proteolytic cleavage of surface proteins in the course of myoblast fusion. The significance of cell surface alterations and the released proteins during the fusion, the involvement of cAMP in the onset of fusion and the possibility that fusion is promoted by external factor(s) are discussed.

• The Korean Journal of Zoology
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• v.31 no.2
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• pp.95-103
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• 1988

Alterations in the amount of fibronectin during chick myogenesis were investigated. The amount of fibronectin as measured by immunoblotting was found to decrease during the process. As a first step in answering the precise mode of change in the level of ibronedin during myogensis, the interaction of 28,000 dalton(28 kDa) amino terminal fragment of fibronectin as well as 85,000 dalton (85 kDa) fragrxient with myoblasts was examined. The specific binding of 125 l-28 kDa fragment to myoblasts was time-dependent and reached a maximum within 60 min. Unlabelled 28 kDa fragment inhibited the binding of 125 I-28 kDa fragment, whereas 85 kDa fragment containing adhesion promoting activity did not inhibit it. This finding suggests that the 28 kDa fragment interacts with the matrix assembly receptors but not with the cell adhesion receptors. Accordingly, the decrease in the level of fibronectin is likely to correlate with the fall of fibronectin receptors on the myoblasts.

• The Korean Journal of Zoology
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• v.25 no.2
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• pp.55-62
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• 1982

DNA synthesis, unscheduled DNA synthesis, excision of pyrimidine dimers and phtoreactivation were determined in UV-irradiated differentiating muscle cells at various times of primary culture of 12 day chick embryos and results obtained were as follows. The rates of UV-induced unscheduled DNA synthesis were increased as increase of UV dose. And the rates were gradually decreased as the increase of time after culture, but at higher doses the decreasing tendency was remarkable. The patterns of DNA replication were changed drastically as a function of time so that in the seven day cultures the rate of $^3$H-thymidine incorporation was found to be 0.2% of the original activity. The pattern of inhibition of DNA replication by UV damage demonstrated that in cells of earlier stages there were no remarkable changes, but in cells of later stages there was significant fluctuation. Photoreactivation and the excision of pyrimidine dimer in the one day cultures showed that photoreactivation occurred immediately after UV-irradiation, but excision of pyrimidine dimer was gradually and slowly occurred. These results indicate that the differentiation of embryonic muscle cells accompanies the gradual reduction of DNA replication and unscheduled DNA synthesis, and that the photoreactivation is rapid process compared to excision repair.

• The Korean Journal of Zoology
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• v.35 no.2
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• pp.161-172
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• 1992

In the short-term treahent of 12-0-tetradecanoylphorbol-13-acetate (TPA) or platelet-derived growth factor (PDGF), the'Wh and PDGF induced the Protein Kinase C (PKC) activation and migration from the cytoplasm to the peripheral nulcear membrane. And the activated PKC which was directly or indirectly stimulated by TPA or PDGF Phosphorylated many kinds of PKC's targeting proteins and induces various biological responses. Especially, the cytoplasmic PKC was phosphorylated within 1 hr and 10 min by TPA-and PDGF-treahent respectivelv. In the long-term treatment of TPA or PDGF, both of them induced the down-regulation and translocation of PKC in the mvoblasts. The down-regulation of PKC isozyrnes, the pattern of PKC I and ll was similar to the PKC 111 isozpnes in the cytoplasm. But in the nucleolus, the TPA did not induce and down-regulation or the inhibition of the immunoreactivity of PKC III antibody. This investigation indicates that each isozvmes of PKC mal be performed the different effects to the down-regulation of the cytoplasm or nucleolus. And douvn-regulated myoblasts contained low immunoreactivity of PKC antibodies.

• The Korean Journal of Zoology
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• v.33 no.2
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• pp.158-165
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• 1990

Proteolytic activity of calpain was found to increase as myoblast fusion proceeds. At 60 hr after cell seeding, lis activity reached to a maximal level and then slighdy decreased thereafter. Similarly, the protein level of calpain reached to a maximal level just proir to the initiation of fusion and remained elevated upon prolonged culture as analyzed by immunoblol using anti-calpain antiserum. These results suggest that the synthesis of calpain is regulated during myogenesis and its proteolytic activity may be related with the process of myoblasts fusion.

• The Korean Journal of Zoology
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• v.29 no.4
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• pp.294-306
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• 1986

In order to find out whether myoblast cells release into the culture medium any substances that induce or promote the fusion of myoblasts, chick embryonic myoblasts were cultured and the cultured medium (muscle-conditioned medium, MCM) was collected. The MCM was then added to the newly cultured myoblasts to examine if it has fusion-promoting activity. The MCM was also analyzed for its protein content before and after its addition to the second culture. The MCM apparently showed fusion-promoting activity when applied to unfused young myoblasts, suggesting that it contained substances that promote the fusion and that had been released from cells fo the previous culture. Analysis of proteins in the myoblasts and in the MCM suggested that the released protein was absorbed by or tightly bound to myoblasts of the second culture. One of the released proteins of about 175 kilodalton was degraded to a polypeptide of approximately 145 kilodalton, which appeared to act upon the membrane proteins of unfused myoblasts so as to stimulate their membrane to fuse with neighboring cells.

• The Korean Journal of Zoology
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• v.35 no.2
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• pp.149-160
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• 1992

Using the protein A-gold complex, the mvoabrillogenesis and actin localization of cultured myoblast were invastisated. In the superstructural changes of mvogenic cell during differentiation, pectoral myoblasts contained large nucleus and numerous ribosomes but no myofibrils during the first 24 hr of cultures. Mvoblast initiated to differentiate at 3-day of culture contained the primitive myofibrillar structure. At 96 hr of culture, the mvofibrillar structure showed reletively discernable Z band but pools defined A, H and M bands. The feature of sarcomeric structure showed more defined form at cultur 5 day. In the aspect of actin localization, actin wvas diffusely detected throughout the cytoplasm of myogenic cell and nucleus during the proliferating stage. At 72 hr of culture, with the appearantc oi primitive mvofibrils, gold particles were observed in surrounding of myofibrils but still presented in overall of cytoplasm, especially in the surface and lumen of endoplasmic reticulum. With the gradual increase of culture time, local distribution of actin was readily detected within cytoplasm. In the 5-day specimen of cultures, gold particles precisely indicate the sites of actin localifation within the sarcomere. These results indicate the time of onset of myofibrill appearance and the biosynthetic and incorporation pathway of actin molecules into sarcomeric structure during myofibrillogenesis. Thus, in the present study, the first mvoabrillar structure was detected at culture 3 day, and the initiation of assembly into a typical sarcmeric structure was observed at culture 5 day. It seems, however, that the course of events on myofibrillogenesis of cultured myoblasts can be changed with great dependence of culture conditions including the number and groluth rate of mononucleated mvoblasts after seeding although the fundamental process shows identical appearances.

• The Korean Journal of Zoology
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• v.35 no.3
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• pp.308-321
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• 1992

심근세포는 골격근 세포와 더불어 세포의 분화기작을 이해하기 위한 좋은 모델이라 할 수 있다. 본 연구에서는 심근세포의 배양중에 일어나는 심근 근원섬유형성과정에 대한 이해를 위하여 SDS- 및 two-dimensional gel electrophoresis와 immunofluorescent 및 immunoelectron microscopy를 수단으로 이를 검토하였다. 배양과정에서 나타나는 주요한 단백질 중에서 SDS-PAGE에 의해 209 kDa와 53 kDa 및 46 kDa band가 각각 막분획 및 세포질 분획 단백질에서 배양과정동안에 가장 현저한 양상을 보였다. 특히 배양 3일과 4일의 양상에서 뚜렷한 변화를 나타내어 이들의 OD 격은 배양 96시간에서의 막분획에서 0.38(209 kDa)와 0.52(53 kDa) 및 배양 72시간에서의 세포질 분획에서 0.34(46 kDa)로 각각 최고치를 나타내었다. 또한 이차원 전기영동상의 양상에서도 세포질 및 막분획 단백질의 전반적인 전개양상이 배양 96시간에서 가장 두드러짐으로써 결국 배양 72시간 및 96시간대에서 심근 모세포의 분화와 관련된 일련의 단백질 합성과정이 가장 활발하다는 것을 알 수 있었다. 배양중에 발현되는 actin에 대한 형광현미경적 분석에 의해, actin은 세포내에서 불균등하게 분포하였고 근원섬유가 형성되는 시기인 배양 96시간에 세포외곽부에서 나타나는 다소 강한 염색성을 관찰할 수 있었다. 심근세포의 근원섬유는 전자현미경 관찰에 의해 primitive forms로 배양 96시간에 출현함을 알 수 있었으며 이 시기의 근원섬유의 형태는 여러 구성대(A, H and M bands)가 미약하나 1대는 비교적 뚜렷하였다. 따라서 심근세포의 T근원섬유형성과정에서 관찰되는 뚜렷한 단백질양상의 변화와 근원섬유의 출현시기 및 구성과정간에는 중요한 관련성이 있으며 이러한 과정에서 actin은 세포질내에서 불균등한 분포를 나타낸다는 것을 알 수 있었다.