Interaction of phospholipid vesicles with cultured myogenic cells : Effects on cell fusion

The effects of defined acyl chain, unilamellar phosphatidylcholine vesicles on the development of cultured embryonic chick muscle was studied. An inhibition of myoblast fusion was observed when vesicles were incubated with cells below the vesicle gel-liquid crystalline phase transition temperature (T_c). This inhibition could be at least partially reversed by culturing the vesicletreated cells above the T_c of vesicles. Evidence supporting adhesion as the mechanism of vesiclecell interaction mediating inhibition of myoblast fusion was derived from scanning electron microscopy (SEM) which demonstrated the presence of vesicle-like particles on the cell membrane under conditions in which myoblast fusion was inhibited. Pretrypsinization of myoblasts before their incubation with vesicles prevented this fusion inhibition, suggesting that vesicles may interact with cell membrane proteins which are involved in the myoblast fusion and/or recognition process.     

Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, β-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.     

乙酰胆碱对培养的人垂体腺瘤细胞增殖的影响

目的：了解乙酰胆碱（acetylcholine,Ach)对人垂体腺瘤细胞增殖的影响。方法：将Ach作用于体外培养的人垂体腺瘤细胞,测定MTT反应A值和^3H-TdR参入量及用流式细胞仪测定细胞周期。结果：10^-7-10^-5mol/LAch可剂量依赖性地使MTT反应A值和^3H-TdR参入量降低,使垂体腺瘤G1期细胞比例增加（P＜0．01）,并可被阿托品阻断。结论：Ach在体外能明显抑制培养的人垂体腺瘤细胞的增殖,这种作用是通过Ach受体来实现的。     

Thyroxine and triiodothyronine influence cultured throid cells: alterations of morphology and mucosubstances.

Further experimentation with the steer thyroid cell line indicates that the formation of "follicles" is enhanced by exposure to 8.9 X 10(-7) M thyroxine (T4) or triiodothyronine (T3) for 96 hr. The production of mucosubstances by the cultured thyroid cells is also increased by treating with T4 or T3, effects demonstrable after staining with PAS-alcian blue at pH 2.5. It is suggested that these in vitro effects bear on morphological organization and synthetic activity of the thyroid gland as well as on autoregulation and intraglandular homeostasis that may occur in situ.     

Sequential study on the influence of adriamycin on cell proliferation and ultrastructure of cultured mammary tumor cells

The time-dependent cytocidal and growth inhibitory effects of Adriamycin (ADM) on monolayer cultures of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor cells were analyzed. The inhibitory effect on cell proliferation was assessed by colony formation in soft agar. Growth inhibition and [3H]thymidine labeling indices clearly demonstrate a dose-dependent antimitotic and cytotoxic effect of the drug. At low concentrations (10(-9)-10(-8) M), 90-100% of cells survived 24-hr exposure. At a higher concentration (10(-5) M), 75-80% of cells survived after 8-hr exposure; by 72 hr only 20-30% of the cells remained. Autoradiographic examination of the pulse-labeled cultures demonstrated no change in the proportion of cells in S-phase during the first 4 hr of treatment. Subsequently DNA synthesis was completely abolished and remained inhibited for the duration of the experiment (72 hr). Clonogenic assay revealed a complete arrest of growth in cells exposed to 10(-5) M ADM and greater than 60% inhibition of cell proliferation at 10(-7) M. Ultrastructural changes were not observed in cells during the first 4 hr of treatment; however, after 8 hr most surviving cells exhibited alterations in nuclear chromatin. The surviving cells showed mitochondrial degeneration, myelin body formation, and vacuolization of the endoplasmic reticulum. This study shows the potential usefulness of the primary culture system in drug evaluation. In addition, serial observation of the effects of ADM revealed a cell subpopulation of the primary culture with differential sensitivity to the drug.     

Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD

Hypoxia alters the biological functions of skeletal muscle cells to proliferate and differentiate into myotubes. However, the cellular responses of myoblasts to hypoxia differ according to the levels of oxygen and the types of cells studied. This study examined the effect of hypoxia (1% oxygen) on bovine satellite cells. Hypoxia significantly increased the proliferation of satellite cells cultured in a growth medium. In addition, the levels of PCNA, cyclin D1, cyclin-dependent kinase-1 (CDK1) and CDK2 expression were increased. Hypoxia facilitated the formation of myotubes as well as the stimulation of MyoD, myogenin, and myosin heavy chain (MHC) expression in differentiating medium (DM) cultures. In particular, satellite cells cultured under hypoxic/DM conditions showed increased p21 expression but not p27. The transfection of satellite cells with antisense MyoD oligonucleotides resulted in a decrease in the MHC, myogenin, MRF4 RNA and protein levels with the concomitant decrease in fused cells to levels similar to those observed under normoxia/DM conditions. This indicates that MyoD up-regulation is closely associated with hypoxia-stimulated myogenic differentiation. In conclusion, hypoxia stimulates the proliferation of satellite cells and promotes their myogenic differentiation with MyoD playing an important role.     

Effect of chemical carcinogens on the ultrastructure of cultured differentiating myogenic cells

The electron microscope studies have been carried out on primary monolayer tissue culture obtained from body tissues of rat and C3H mouse embryos. The cells of tissue culture were mainly myoblasts and fibroblast-like cells. The cultures were treated with two different carcinogenic substances--benz(a)-pyrene (BP) and methylnitrosonitroguanidine (MNNG). The changes were uniform and showed some alterations in formation of cell complexes, the inhibition of development and maturing of muscle elements, and distrophy of cytoplasmic organelles. The revealed distinction in morphological reaction of myogenic cells to the effect of BP and MNNG were wave-like myofibrillar structures in MNNG-treated cultures.     

Effect of bone marrow cells on colony-forming stromal cells in guinea pigs and on the proliferation of their cultured descendents

In the presence of irradiated bone marrow cells the efficiency of stromal colony formation increases from 0.8 +/- 0.2 to 3.6 +/- 0.4 per 10(4) explanted bone marrow cells. The growth-stimulating activity of bone marrow cells on passaged bone marrow fibroblasts depends on growth conditions of passages to which irradiated bone marrow cells are added. The response of proliferating bone marrow fibroblasts to stimulating activity of bone marrow cells is low, while addition of bone marrow cells to fibroblast cultures stimulates their proliferation.     

Effects of gender-specific adult bovine serum on myogenic satellite cell proliferation, differentiation and lipid accumulation

The study was performed to explore the effects of adult bovine male serum (MS), female serum (FS), and castrated male serum (C-MS) on myogenic satellite cells (MSCs) proliferation and differentiation into myotubes or into adipocyte-like cells (ALCs). MSC proliferation and differentiation was highest in the medium supplemented with MS, implying the important role of male steroid hormones. Myogenin and desmin were highly upregulated in cells cultured in MS-supplemented medium. In contrast, lipid accumulation in ALCs was highest in the medium supplemented with FS. Fatty acid transporter (FAT/CD36) was upregulated in FS-supplemented cultures. Detection of higher FAT/CD36 inducing fatty acids (arachidic acid and eicosapentaenoic acid) in FS compared with MS and C-MS suggests that these fatty acids may have influenced the enhanced formation of lipid droplets in ALCs. Effect of sex steroids on cell proliferation and cell growth of bovine MSCs and C2C12 cell in C-MS was greater than charcoal-dextran-treated fetal bovine serum (CDFBS). Concluding the above facts, the results indicate that each gender-specific bovine serum constitutes of different component, which leads to unique effects on cell behavior.     

In vitro proliferation and differentiation of myogenic cells from adult Xenopus

A technique is described for isolating amphibian myogenic cells from the muscle of adult Xenopus laevis (Dauchin). Muscles were dissociated with 0.2% collagenase and 0.1% trypsin. The resulting cell suspensions were separated from the remaining myofibres by filtration through nylon grids. Most of the cells remaining in the filtrate suspension were satellite cells or fibroblasts. When plated in Petri dishes, satellite cells adhered to the substrate, became spindle-shaped and proliferated activity in a culture medium supplemented with fetal calf serum. Mitotic waves lasted 4 days and consequently cell density markedly increased. Satellite cells came into contact and began to fuse into myotubes on day 8 of culture. Horse serum, which replaced fetal calf serum in the medium on day 12, accelerated cell fusions which were almost complete on day 18. However, under these conditions, some mononucleated cells continued to undergo mitosis. Cell proliferation with a high rate of mitosis was prolonged by repeated trypsinization and replating in medium supplemented with fetal calf serum. When myofibres from dissociated muscles were cultured under the same conditions, they never fragmented or divided.     

Decorin enhances the proliferation and differentiation of myogenic cells through suppressing myostatin activity

Decorin, a small leucine-rich proteoglycan, plays an important role in the regulation of cell growth. Our recent study has shown that immobilized decorin in the collagen matrix sequesters myostatin into the extracellular matrix and prevents its inhibitory action to myoblast proliferation in vitro. However, it still remains unclear whether free decorin could affect the proliferation and differentiation of myogenic cells by regulating myostatin activity. In the present study, we generated stable clonal C2C12 myoblasts that were over-expressing decorin, and showed that decorin over-expressing cells had an increased rate of proliferation as compared to control cells. Decorin over-expressing cells formed multi-giant hypertrophic myotubes with an elongated morphology and larger size as compared to control cells, although the initiation of differentiation in decorin over-expressing cells was somewhat delayed as compared to control cells. Western blot analysis demonstrated that MyoD expression in decorin over-expressing cells was lower than that in control cells until 12 h after induction to differentiate. At 48-h differentiation, the expressions of MyoD, p21 and myogenin were dramatically increased in cells that over-expressed decorin. Furthermore, we revealed that over-expression of decorin suppressed the activity of myostatin endogenously synthesized in C2C12 myoblasts and attenuated the signaling of exogenous myostatin. Consistent with these results, knock-down of decorin impairs C2C12 myoblast growth by increasing the sensitivity to exogenous myostatin. These results clearly show that decorin enhances the proliferation and differentiation of C2C12 myoblasts through suppressing myostatin activity.     

Effect of porcine placenta steroid extract on myogenic satellite cell proliferation, transdifferentiation, and lipid accumulation

Intramuscular long-chain fatty acids (LCFAs) play an important role in energy production and initiation of mitochondrial oxidation of lipids. Herein, we report a natural porcine placenta steroid extract (PPSE) that stimulates transdifferentiation and lipid accumulation in bovine myogenic satellite cells (MSCs). The steroids hormones in PPSE were analyzed using enzyme-linked immunosorbant assay and presence of LCFA was established using gas chromatography. At 70% confluent growth, cells were treated with PPSE, LCFAs, transdifferentiation cocktail and commercially available steroid hormones. The working concentrations of all chemicals were manipulated similar to PPSE. The cells were observed for morphological changes and subjected to quantitative analysis of lipid deposition on Days 2, 4, and 6 of treatment. PPSE-treated MSCs exclusively transformed into lipid-accumulated adipose-like cells (ALCs). However, myotubes or adipocytes were formed in cells treated with other chemicals. Expression of different genes was studied to ascertain the molecular mechanism involved in ALC formation. CD36, fatty acid binding protein 4, and peroxisome proliferator-activated receptor-gamma were up-regulated. The expression of CD36 was established through immunocyto-chemical analysis. A viability assay was used to confirm the effect of PPSE on proliferation of MSCs. Hence, a natural steroid extract from porcine was found as a nontoxic mixture, which induces lipid accumulation and transdifferentiation of MSCs to ALCs. From the gene expression studies, it was established that the extract works almost in homogenous manner with other lipid inducers.     

Hypertriglyceridemic serum from magnesium-deficient rats induces proliferation and lipid accumulation in cultured vascular smooth muscle cells

An important characteristic of hyperlipemia associated with magnesium deficiency in rats is the postprandial accumulation of triglyceride-rich lipoproteins. The present investigation was performed to determine the effect of serum from magnesium-deficient animals on cultured vascular smooth muscle cells (VSMC). Sera were obtained from control and magnesium-deficient rats fed adequate or deficient diets for 8 days. Magnesium-deficient animals were hypertriglyceridemic compared with control rats, but their total cholesterolemia was not significantly modified. Pooled sera from control and magnesium-deficient animals were added to the culture medium at various concentrations. The maximum of proliferation for both control and magnesium-deficient sera was reached when they were added at 6% to the culture medium and on day 4 after the begining of incubation. Medium containing serum from magnesium-deficient rats stimulated the cell proliferation as monitored by cell count and [³H]thymidine incorporation. Staining of VSMC with Oil red O and measuring lipids have shown a marked lipid accumulation (triglycerides) in cells incubated with serum obtained from magnesium-deficient animals compared with serum from control rats. These results indicate that serum from magnesium-deficient rats contains factors that stimulate proliferation of arterial medial cells and that hyperlipemia associated with magnesium-deficiency may cause lipid accumulation in vascular cells.     

Motility,linear arrangement and cell-to-cell contact of myogenic cells prior to fusion

Summary Time-lapse cinematography elucidates the genesis of a uniform and approximately linearly arranged myogenic cell aggregate, stemming from two larger cell groups. The ultimate aggregate is created by continuous movement of one cell group toward the other. Following this motion, the angle between the cell groups is reduced as they approach each other.Different patterns of cell motility can be recognized. Some cells move in a preferred direction in relation to the aggregate as a whole, whereas others alter their direction of movement.The myogenic cells are aligned end-to-end and side-by-side. The latter is often accomplished in the following manner: two cells in end-to-end contact form as crescent-shaped free space with their polar extensions; a neighboring spindle-shaped cell then settles in this space. An arrangement of cells such that their greatest cytoplasmic widths lie at the same level can also be seen. During the recording period, two cells in one of the groups were replicating. One of them realized karyo- and cytokinesis in approximately 80min. The daughter cells moved apart in opposite directions, but never lost contact to the aggregate. This observation shows that contact between presumptive myoblasts and myoblasts is established.This research was supported by a grant from the Deutsche Forschungsgemeinschaft (Ba 689/1)     

Cell fusion and differentiation in cultured chick muscle cells

A method for the quantitative estimation of myoblast fusion and the effects of variables in the culture conditions on the extent of fusion are described. The onset of fusion is delayed both by feeding and by lowering the initial cell density, but effects of alterations in sera and embryo extracts are evident only at later stages of differentiation. Using a sensitive fluorimetric assay for creatine phosphokinase, enzyme activity was determined throughout the culture period and related to the extent of cell fusion. The increase in activity during a 7-day culture period is biphasic, the first small increase (2–5 ×), which begins after 40–50 h in culture, being apparently closely related to cell fusion. There is a 48 h delay before the second, much larger (up to 25 ×) increase begins.     

Phospholipids of membranes of cultured cells and the products of protoplast fusion

The variety and distribution of phospholipids in the cell membranes of cultured cells and their fatty acid composition were analysed. Membranes of suspension cultured cells of Rauwolfia serpentina var. Bentham, Nicotiana tabacum var. Samsun, Atropa belladonna and Bouvardia ternifolia had almost the same composition of phosphatidylcholine, PC (ca 50%); phosphatidylethanolamine, PE (ca 25%); phosphatidylinositol, PI (ca 10%); phosphatidylglycerol, PG (several %); and phosphatidic acid, PA (several %). We determined the distribution of the molecular species of the three major phospholipid fractions (PC, PE and PI) in R.serpentina and N.tabacum. Membranes of both cell-types contained basically similar molecular species, 1–16:0/2–18:2 the main type, particularly in the PC- and PE- fractions; 1–18:2/2–18:2 and 1–18:0/2–18:2 for R. serpentina; and 1–16:0/2–18:3, 1-18:0/2-18:3 and 1-18:2/2-18:2 for N.tabacum. The influence of membrane fluidity on protoplast fusion as effected by the phase transition of the phospholipids, is discussed.     

Effect of transforming growth factor beta on proliferation of L6 and embryonic porcine myogenic cells

We have examined the effect of Transforming Growth Factor (TGF) beta on proliferation of L6 and embryonic porcine myogenic cells. Proliferation of L6 cells was suppressed by both TGF beta-1 and TGF beta-2 in a dose-dependent manner. Half-maximal suppression of proliferation occurred at .036 ng TGF beta-1/ml and .06 ng TGF beta-2/ml. Maximal inhibition (60% suppression of proliferation for TGF beta-1 and 52% for TGF beta-2) occurred between .1 and .3 ng/ml for each growth factor. Suppression of proliferation was completely abolished in the presence of an anti-TGF beta antibody that inhibited the biological activity of TGF beta-1 and TGF beta-2. When we evaluated the effect of TGF beta-1 on proliferation of embryonic porcine myogenic cells we obtained results which were very similar to those obtained for L6 cells. Insulin-like growth factor (IGF)-I stimulated proliferation of L6 cells in a dose-dependent manner in serum-free, defined medium. However as little as .02 ng TGF beta-1/ml detectably suppressed this stimulation and .3 ng TGF beta-1/ml caused a 60% reduction in cell number in cultures treated with 30 ng IGF-l/ml. Thus TGF beta-1 significantly suppressed IGF-I-stimulated proliferation of L6 cells.