Analysis of the expression of chicken and rat gene products in myoblast x myoblast cell hybrids

Hybrid cells were isolated by fusing primary chicken myoblasts to HPRT-deficient rat L6 myoblasts and incubating the cells in medium containing HAT and ouabain. All hybrid clones contained both rat and chicken chromosomes and expressed a number of gene products characteristic of both species. Although all clones were capable of fusing spontaneously to form myofibers, immunofluorescence and isoenzyme analysis revealed only the rat forms of skeletal muscle myosin and MM-creatine kinase. No differentiated gene products of chicken origin were detected. Analysis of the expression of chicken HPRT revealed that some hybrid clones were capable of modulating this enzyme activity when switched from HAT medium into thioguanine medium and back into HAT, even though HPRT is normally a constitutively expressed enzyme. Parental control cells were incapable of this modulation phenomenon.     

Analysis of the expression of chicken and rat gene products in myoblast × myoblast cell hybrids

Hybrid cells were isolated by fusing primary chicken myoblasts to HPRT-deficient rat L6 myoblasts and incubating the cells in medium containing HAT and ouabain. All hybrid clones contained both rat and chicken chromosomes and expressed a number of gene products characteristic of both species. Although all clones were capable of fusing spontaneously to form myofibers, immunofluorescence and isoenzyme analysis revealed only the rat forms of skeletal muscle myosin and MM-creatine kinase. No differentiated gene products of chicken origin were detected. Analysis of the expression of chicken HPRT revealed that some hybrid clones were capable of modulating this enzyme activity when switched from HAT medium into thioguanine medium and back into HAT, even though HPRT is normally a constitutively expressed enzyme. Parental control cells were incapable of this modulation phenomenon.     

Intracellular antigen migration in interspecific myoblast heterokaryons

Intracellular migration of species-specific nuclear antigens was studied in chick-rat heterokaryons. These cells were produced by virus-induced or spontaneous fusion of different chick cells with rat myoblasts or myotubes. Chick erythrocyte nuclei introduced into rat myogenic cells increased in volume and were reactivated to synthesize RNA. As the chick erythrocyte nuclei enlarged, they rapidly accumulated rat nuclear antigens. Rat nucleolar and nucleoplasmic antigens assumed a distribution in the chick nuclei corresponding to that in rat nuclei. In hybrid myotubes formed by the spontaneous fusion of chick myoblasts and rat myoblasts antigen exchange was at a much lower level. Some exchange of both rat and chick nuclear antigens could, however, be detected also in this system. Thus chick nuclear envelope and nucleolar antigens migrated into the rat myoblast nuclei and assumed an intranuclear localization analogous to that in chick nuclei. On the basis of these results it appears that antigenic nuclear macromolecules are constantly exchanged between the rat and chick nuclear compartments and the cytoplasm of the heterokaryon. During the rapid nuclear swelling which occurs when chick erythrocyte nuclei are activated in rat myoblast heterokaryons, the inward migration of rat nuclear antigens into the chick erythrocyte nucleus is more impressive than the migration of chick antigens into the rat nuclei.     

Water on hydrophobic surfaces: mechanistic modeling of polyethylene glycol-induced protein precipitation

Bioprocess and Biosystems Engineering - For the purification of biopharmaceutical proteins, liquid chromatography is still the gold standard. Especially with increasing product titers, drawbacks...     

Phenotypic expression in chick erythrocyte X rat myoblast hybrids and in chick myoblast X rat myoblast hybrids

Attempts were made to reprogram chick erythrocyte nuclei to specify the synthesis of chick myosin. Chick erythrocytes were fused with rat myogenic cells with the aid of UV-inactivated Sendai virus. In the heterokaryons and hybrid myotubes which resulted from this fusion, the erythrocyte nuclei resumed RNA synthesis and formed nucleoli. Although some new chick antigens developed in those myotubes which contained fully reactivated chick erythrocyte nuclei, accumulation of chick myosin could not be detected by immunological methods. Neither small heterokaryons nor large hybrid myotubes which were actively synthesizing rat myosin reacted with antibodies directed against chick myosin. A small number of mononucleated cells, believed to be synkaryons formed by mitotic division of heterokaryons, did, however, react strongly with antibodies directed against chick myosin and showed a cross striation typical of skeletal muscle. The frequency of such cells was too low, however, to permit karyological analysis or further characterization of the antigen. Hybrids between chick myoblasts and rat myoblasts produced both chick and rat myosin thus indicating that simultaneous translation of chick and rat mRNA for myosin in a common cytoplasm was possible. In summary the evidence obtained suggested that reprogramming of chick erythrocyte nuclei, if it did occur in the present system, was a rare phenomenon.The possibility that hybrids between chick erythrocytes and rat myoblasts expressed markers typical of an erythroid phenotype was examined by immune staining with antibodies directed against chick haemoglobin. The results suggested that haemoglobin was introduced into hybrid cells by erythrocytes which failed to lyse before fusion. The intensity of this immune fluorescence decreased with increasing time after fusion. The rate at which this decrease occurred was not affected by inhibition of RNA synthesis. Thus, there was no evidence for the accumulation of haemoglobin in the hybrid cells.     

Control of differentiation in heterokaryons and hybrids involving differentiation-defective myoblast variants

Clones of differentiation-defective myoblasts were isolated by selecting clones of L6 rat myoblasts that did not form myotubes under differentiation-stimulating conditions. Rat skeletal myosin light chain synthesis was induced in heterokaryons formed by fusing these defective myoblasts to differentiated chick skeletal myocytes. This indicates that the structural gene for this muscle protein was still responsive to chick inducing factors and that the defective myoblasts were not producing large quantities of molecules that dominantly suppressed the expression of differentiated functions. The regulation of the decision to differentiate was then examined in hybrids between differentiation- defective myoblasts and differentiation-competent myoblasts. Staining with antimyosin antibodies showed that the defective myoblasts and homotypic hybrids formed by fusing defective myoblasts to themselves could in fact differentiate, but did so more than a thousand times less frequently than the 64% differentiation achieved by competent L6 myoblasts or homotypic competent X competent L6 hybrids. Heterotypic hybrids between differentiation-defective myoblasts and competent L6 cells exhibited an intermediate behavior of approximately 1% differentiation. A theoretical model for the regulation of the commitment to terminal differentiation is proposed that could explain these results by invoking the need to achieve threshold levels of secondary inducing molecules in response to differentiation-stimulating conditions. This model helps explain many of the stochastic aspects of cell differentiation.     

Hypoxia affects mitochondrial protein expression in rat skeletal muscle

Hypoxia affects mammalian mitochondrial function, as well as mitochondria-based energy metabolism. The detail mechanism has not been fully understood. In this study, we detected protein expression levels in mitochondrial fractions of Wistar rats exposed to hypobaric hypoxia by use of proteomic methods. Adult male Wistar rats were randomized into an hypoxic (4,500?m, 30 days) group and a normoxic control group (sea level). Gastrocnemius muscles mitochondria were extracted and purified. Mitochondrial oxygen consumption was measured with a Clark oxygen electrode; mitochondrial transmembrane potential was detected with Rhodamine 123 as a fluoresce probe. Using 2-DE and MALDI-TOF MS analysis, we identified eight mitochondrial protein spots that were differentially expressed in the hypoxic group compared with the normoxic control. These proteins included Chain A of F1-ATPase, voltage dependent anion channel 1 (VDAC), hydroxyacyl Coenzyme A dehydrogenase α-subunit, mitochondrial F1 complex γ-subunit, androgen-regulated protein and tripartite motif protein 50. Two of the spots, VDAC and ATP synthase α-subunit, were confirmed by Western blotting analysis. Oxygen consumption during State 3 respiration, as well as the respiratory control ratio (RCR) was significantly higher in the control than that in the hypoxic group; mitochondrial transmembrane potential was significantly higher in hypoxic group than that in the control. With successful use of multiple proteomic analysis techniques, we demonstrates that 30 days hypoxia exposure has effects on the expression of mitochondrial proteins involved in ATP production and lipid metabolism, decrease the stability of mitochondrial membrane, and affect the mitochondrial electron transport chain.     

The mechanism of polyethylene glycol-induced natriuresis in rats: role of atrial natriuretic hormone

A putative role of atrial natriuretic hormone (ANH) in a polyethylene glycol (PEG) 200-induced natriuresis was examined in conscious Wistar rats. Low molecular weight PEG 200 (0.5 or 1.0 ml/100g body weight) was orally administered to rats by gavage. Urine was collected during a 3 hr test period and blood was obtained at the end of each experiment for measurement of ANH, PRA, clearance studies and for indirect indices of plasma volume. Urinary excretion of sodium and volume increased while plasma ANH concentrations were markedly decreased in a dose-related manner following PEG 200 administration. The osmotic clearance was also elevated following PEG 200 administration. No significant change was observed in any of the parameters following high molecular weight PEG 8000. The observed decrease in ANH was associated with an apparent contraction of plasma volume despite the increased serum osmolality. These data indicate that the ANH inhibitory influence of the decreased plasma volume takes precedence over the stimulatory effect of the hyperosmolality and the latter is primarily responsible for the increased osmotic clearance and natriuresis observed in this model.     

Cytogenetic characteristics of 26 polyethylene glycol-induced human-hamster hybrid cell lines

A cytological analysis of 26 polyethylene glycol (PEG) induced human/hamster hybrid lines has shown that such lines are similar to inactivated Sendai virus (ISV) induced hybrids in respect to stability, retention of specific chromosomes, and cell selection. The evolution of stable hybrid cell lines carrying variable human chromosome complements depends upon a balance being established between the retained human and hamster genomes. This balance is a result of random loss of human and hamster chromosomes followed by selection of the fittest stem lines. A major mechanism ofchromosome loss may be fragmentation and elimination of acentric fragments. Twelve of the 26 lines had stabilized by the 30th passage, an incidence similar to that found with ISV-induced hybrids studied in this laboratory. Thus, PEG may be considered to be an ideal chemical for inducing somatic cell hybrids for genetic analysis.     

The proteome of chicken skeletal muscle: changes in soluble protein expression during growth in a layer strain

The whole animal, and the pectoralis muscle in particular, grows at a greatly enhanced rate in chickens selected for meat production (broilers) when compared to those selected for egg production (layers). As part of an ongoing study to analyse muscle protein dynamics under conditions of rapid growth, we have embarked upon a preliminary characterisation of the proteome of layer chicken pectoralis muscle, at specified time-points from 1 to 27 days after hatching. Soluble extracts of muscle homogenates were separated by two-dimensional (2-D) gel electrophoresis and selected spots were analysed by in-gel tryptic digestion and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Of 90 spots, 51 gave mass spectra that matched to existing chicken proteins present in on-line databases, 12 matched equivalent proteins from non-avian species and 11 yielded good quality spectra but were unable to be matched against existing databases. For many of these proteins, growth over 27 days elicited dramatic changes in relative expression levels. Chicken skeletal muscle offers an excellent system for developmental proteomics.     

F-actin is involved in the polyethylene glycol-induced fusion of chick embryo fibroblasts

Cytochalasin B inhibits the polyethylene glycol (PEG)-induced fusion of chick embryo fibroblasts. Induction of fusion of these cells by PEG is associated with transient changes in the pattern of F-actin organization within the cell. The changes include the disappearance of stress fibres and accumulation of F-actin under the plasma membrane. These results suggest an involvement of F-actin in PEG-induced cell fusion.     

Ascorbic acid facilitates chicken myoblast fusion in vitro

Summary Addition of low concentrations of ascorbic acid (5 μg/ml to 10 μg/ml) to myogenic chick embryo cultures resulted in an early fusion. At 30 h cultures receiving small amounts of ascorbic acid presented fusion rates 3 times that of the control. However, control rates of fusion were not different from those of experimentals at 50 h. No such effect was seen with ascorbic acid added at 24 h of culture, or with ascorbic acid addition to a calcium-deprived system. These findings demonstrate that the calcium binding properties of ascorbic acid can induce precocious myogenic fusion.     

Effect of high density lipoproteins on protein expression in myoblast cell lines

We have studied the effects of high-density lipoprotein (HDL) on rat heart (H9c2) and skeletal (L6) myoblasts using two-dimensional gel electrophoresis and tandem mass spectrometry. Gels generated from control cells and cells treated with 250 microg/mL HDL showed significant differences in the 7-10 pI region and the 30-50 kDa mass region. In particular, the membrane binding protein, annexin II, the voltage-dependent anion channel, glyceraldehyde-3-phosphate dehydrogenase and other glycolytic proteins are differentially expressed.     

Differential expression of an endogenous mannose-binding protein R1 during muscle development and regeneration delineating its role in myoblast fusion

The role of the endogenous brain carbohydrate-binding proteinR1 in muscle cell development and regeneration was analysedboth in vivo and in vitro. In vivo, R1 was developmentally regulated,with an embryonic 65 000 subunit and a neonatal 67 000 subunit,being replaced progressively by a 135 000 adult form. LectinR1 was intracellularly localized at birth and in the prenatalperiod. During development and at the time of myoblast fusion,the antigen was progressively found at the surface, where itremained at low levels in the adult. In vitro, in pure myoblastcultures, only the embryonic form was present. The ultrastructuralstudies indicated that the lectin could participate in the membranefusion process during myoblast fusion. The specific role inmyoblast fusion, derived from the ultrastructural localizationof R1, was evidenced by a strong inhibitory effect of anti-R1 Fab fragments (10–100 µg/ml), relative to controlFab fragments. In vivo, the embryonic subunit pattern and subcellulardistribution of R1 reappeared in muscle cells after lesion ofthe adult muscle. This suggested that, as observed in vitro,R1 participated in vivo in the phenomenon of myoblast fusion.Similar modifications in subunit expression were observed inmuscles after denemation (the embryonic form of lectin R1 reappearingafter lesion), suggesting that R1 could be involved in the processof neuromuscular junction formation. Thus, it is proposed thatthe carbohydrate-binding protein R1 is an important recognitionmolecule for the formation of myotubes. Its potential involvementin a recognition process between axons and muscle cells duringneuromuscular junction formation is discussed. culture development fusion N-glycan glycoprotein lectin mannose myoblast     

Polyethylene glycol-induced mammalian cell hybridization: effect of polyethylene glycol molecular weight and concentration.

The effects of polyethylene glycol (PEG) molecular weight and concentration on mammalian cell hybridization were studied. The peak hybridization-inducing activity with all grades of PEG from 400-6000 was found to occur in the concentration range of 50-55%. However, changes in concentration were seen to have different quantitative effects with different grades of PEG. For monolayer fusions, PEG 1000 at 50% seems to be the optimal combination of PEG molecular weight and concentration, in terms of both efficiency of hybridization and relative insensitivity to dilution effects.