scholarly journals Turnover of the creatine kinase subunits in chicken myogenic cell cultures and in fibroblasts

1981 ◽  
Vol 196 (2) ◽  
pp. 377-382 ◽  
Author(s):  
M Caravatti ◽  
J C Perriard
Keyword(s):  
Creatine Kinase ◽  
Myosin Heavy Chain ◽  
Cell Cultures ◽  
Heavy Chain ◽  
Half Life ◽  
Regulatory Mechanism ◽  
Total Acid ◽  
Myogenic Cells ◽  
Creatine Kinase Isoenzyme ◽  
The Stability

The rates of degradation of creatine kinase subunits, M-CK and B-CK subunits, were measured in cultured myogenic cells and in subcultured fibroblasts. In differentiated myogenic cells, the myotubes, both M-CK and B-CK subunits are synthesized. Their rates of degradation were compared. The M-CK subunits is slightly more stable and is degraded with an average apparent half-life of 75 h, whereas that of the B-CK subunit was shorter with 63 h. The turnover properties of M-CK subunit from soluble and of myofibril-bound MM-CK homodimeric creatine kinase isoenzyme isolated from breast muscle of young chickens were identical. The apparent half-life of the B-CK subunit was also determined in subcultured fibroblasts and 5-bromo-2′-deoxyuridine-treated cells, and found to be shorter than in myotubes (46 h and 37 h respectively). Similar observations were made for myosin heavy chain, actin and total acid-precipitable material. It appears therefore that proteins are in general degraded more slowly in differentiated myogenic cells. The differences in the stability of M-CK and B-CK subunits in myotubes probably do not reflect a major regulatory mechanism of the creatine kinase isoenzyme transition.

Myogenic cell migration from somites is induced by tissue contact with medial region of the presumptive limb mesoderm in chick embryos

Development ◽  
1995 ◽  
Vol 121 (3) ◽  
pp. 661-669 ◽  
Author(s):  
K. Hayashi ◽  
E. Ozawa
Keyword(s):  
Cell Migration ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Flank Region ◽  
Myogenic Cell ◽  
Chick Embryos ◽  
Medial Region ◽  
Myogenic Cells ◽  
Limb Buds ◽  
Lateral Region

It is known that myogenic cells in limb buds are derived from somites. In order to examine the potential of the limb primordium (presumptive limb somatopleure) to induce myogenic cell migration, we transplanted chick presumptive limb somatopleure to the flank region of an embryo, a region that does not normally contribute myogenic cells to the limb. Somitic cell migration was examined using a vital labeling technique. When the presumptive limb somatopleure was transplanted and was in contact with the host flank somite, somitic-cell migration toward the graft was observed. The labeled somitic cells within the graft were identified as myogenic cells in two ways: first, we found that N-cadherin-expressing cells appeared in the graft. Second, after 3 further days of incubation, the somitic cells formed dorsal and ventral masses and expressed sarcomeric myosin heavy chain within the graft. Cell migration occurred only when the somite was in contact with the medial region of the presumptive limb somatopleure. When the somite was not in contact with the limb somatopleure, or when the somite was in contact with the lateral region of the limb somatopleure, migration did not occur. These observations indicate that the potential to induce myogenic cell migration is restricted to the medial region of the presumptive limb somatopleure and that tissue contact is required.

Adaptive changes in cardiac myosin heavy chain and creatine kinase isozymic profiles in rats native of altitude

2005 ◽  
Vol 184 (2) ◽  
pp. 95-104 ◽  
Author(s):  
A. Letout ◽  
M. Solares-Espinoza ◽  
P. Mateo ◽  
N. Koulmann ◽  
L. Bahi ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Cardiac Myosin ◽  
Adaptive Changes

Myosin heavy chain expression in embryonic cardiac cell cultures

1986 ◽  
Vol 115 (1) ◽  
pp. 204-214 ◽  
Author(s):  
B.J. Zadeh ◽  
A. González-Sánchez ◽  
D.A. Fischman ◽  
D.M. Bader
Keyword(s):  
Myosin Heavy Chain ◽  
Cell Cultures ◽  
Heavy Chain ◽  
Cardiac Cell ◽  
Cardiac Cell Cultures

scholarly journals The stability of envelope-pseudotyped lentiviral vectors

Gene Therapy ◽  
2020 ◽  
Author(s):  
Iris J. C. Dautzenberg ◽  
Martijn J. W. E. Rabelink ◽  
Rob C. Hoeben
Keyword(s):  
Cell Cultures ◽  
Half Life ◽  
Mammalian Cells ◽  
Culture Medium ◽  
Lentiviral Vector ◽  
Virus Titer ◽  
Lentiviral Vectors ◽  
Envelope Proteins ◽  
The Stability ◽  
Vector Particles

Abstract Lentiviral vectors have become popular tools for stable genetic modification of mammalian cells. In some applications of lentiviral vector-transduced cells, infectious-lentiviral particles should be absent. Quantification of the free-vector particles that remain from the inoculum can be difficult. Therefore a formula was established that yields an estimation of the ‘Reduction Ratio.’ This ratio represents the loss of titer based on a number of vector-inactivating effects. In this study, we evaluated several parameters and assumptions that were used in the current formula. We generated new data on the stability and trypsin sensitivity of lentiviral vectors pseudotyped with eight heterologous envelope proteins and the loss of vectors by washing or passaging the cell cultures. Our data demonstrate that the loss of virus titer under the influence of trypsin as well as the half-life of the particles in tissue culture medium is dependent on the vector’s envelope protein. While VSV-G-envelope-pseudotyped particles were unsensitive to trypsin, the titer of vectors pseudotyped with other envelope proteins decreased 2–110-fold. The half-life in culture medium ranged from 8 to 40 h for the different envelope-pseudotyped vectors, with 35 h for VSV-G-envelope-pseudotyped vector particles. Additionally, we found that removal of the culture medium from Ø35 mm to Ø10 cm dishes reduces the amount of vector particles in the culture by 50-fold and 20-fold, respectively. Together these data can be used to more precisely estimate the maximum number of free lentiviral vector particles in cell cultures.

Identification of a novel cardiac-specific transcript critical for cardiac myocyte differentiation

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2779-2789 ◽  
Author(s):  
Y. Wei ◽  
D. Bader ◽  
J. Litvin
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Cardiac Myocyte ◽  
Heart Tube ◽  
Myogenic Cells ◽  
Chicken Heart ◽  
Reading Frame ◽  
Cardiac Myogenesis ◽  
Myocyte Differentiation ◽  
Heart Formation

A novel cDNA, pCMF1, which is expressed exclusively and transiently in the myogenic cells of the differentiating chicken heart was isolated and characterized. The full-length cDNA of pCMF1 has one open reading frame encoding 1538 predicted amino acids. While computer analysis predicts the presence of specific structural motifs, the overall sequence of pCMF1 is unique. The pattern of pCMF1 gene expression during heart formation was determined by whole-mount in situ hybridization. pCMF1 is transiently expressed within the myogenic cells of the primitive heart tube from stages 9 to 18 and is not detected in the heart or any other tissue thereafter. A replication-deficient retrovirus was used to mediate pCMF1 antisense expression in cardiogenic mesoderm. These analyses determined that the presence of pCMF1 antisense sequences disrupted myosin heavy chain expression during cardiac mesoderm differentiation. pCMF1 antisense had no effect on myosin heavy chain expression in differentiated cardiac myocytes. These data suggest a potential function for pCMF1 during cardiac myogenesis.

scholarly journals MyoD, myogenin independent differentiation of primordial myoblasts in mouse somites.

1992 ◽  
Vol 116 (5) ◽  
pp. 1243-1255 ◽  
Author(s):  
M G Cusella-De Angelis ◽  
G Lyons ◽  
C Sonnino ◽  
L De Angelis ◽  
E Vivarelli ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Posttranscriptional Regulation ◽  
Proximal Region ◽  
Limb Bud ◽  
Northern Analysis ◽  
Myogenic Cells

The accumulation of two myogenic regulatory proteins, MyoD and myogenin, was investigated by double-immunocytochemistry and correlated with myosin heavy chain expression in different classes of myoblasts in culture and during early myogenesis in vivo. During in vitro differentiation of fetal myoblasts, MyoD-positive cells were detected first, followed by the appearance of cells positive for both MyoD and myogenin and finally by the appearance of differentiated myocytes and myotubes expressing myosin heavy chain (MHC). A similar pattern of expression was observed in cultures of embryonic and satellite cells. In contrast, most myogenic cells isolated from newly formed somites, expressed MHC in the absence of detectable levels of myogenin or MyoD. In vivo, the appearance of both myogenin and MyoD proteins was only detected at 10.5 d postcoitum (d.p.c.), when terminally differentiated muscle cells could already be identified in the myotome. Parasagittal sections of the caudal myotomes of 10.5-d-old embryos showed that expression of contractile proteins preceded the expression of myogenin or MyoD and, when coexpressed, MHC and myogenin did not co-localize within all the cells of the myotome. In the limb bud, however, many myogenin (or MyoD) positive/MHC negative cells could be observed in the proximal region at day 11. During further embryonic development the expression of these proteins remained constant in all the muscle anlagens examined, decreasing to a low level during the late fetal period. Western and Northern analysis confirmed that the myogenin protein could only be detected after 10.5 d.p.c. while the corresponding message was clearly present at 9.5 d.p.c., strongly suggesting a posttranscriptional regulation of myogenin during this stage of embryonic development. These data show that the first myogenic cells which appear in the mouse myotome, and can be cultured from it, accumulate muscle structural proteins in their cytoplasm without expressing detectable levels of myogenin protein (although the message is clearly accumulated). Neither MyoD message or protein are detectable in these cells, which may represent a distinct myogenic population whose role in development remains to be established.

scholarly journals Fixation and immunofluorescent analysis of creatine kinase isozymes in embryonic skeletal muscle.

1987 ◽  
Vol 35 (7) ◽  
pp. 717-722 ◽  
Author(s):  
M M Robinson
Keyword(s):  
Skeletal Muscle ◽  
Creatine Kinase ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Fixation Procedure ◽  
Creatine Kinase B ◽  
Immunofluorescent Analysis ◽  
Embryonic Muscle ◽  
Creatine Kinase Isozymes

Pectoral muscles from chicken embryos of various ages were examined with immunofluorescent and radiolabeled probes for the presence of brain-type creatine kinase (B-CK), muscle-specific creatine kinase (M-CK), muscle-specific myosin heavy chain (MHC), and cycling cells. The diffusible creatine kinase isozymes were not detectable by indirect immunofluorescence after standard histological fixation of embryonic muscle. However, a fixation procedure was devised that permitted immunodetection of the creatine kinase isozymes (particularly B-CK) in embryonic tissue from all stages of development studied. B-CK, M-CK, and MHC were all detected in post-mitotic muscle cells, but only B-CK was detected in cycling cells. Correlations between these findings and in vitro observations of a deterministic muscle lineage are discussed.

Stability of commonly used thiols and of human creatine kinase isoenzymes during storage at various temperature in various media.

1977 ◽  
Vol 23 (5) ◽  
pp. 816-829 ◽  
Author(s):  
D A Nealon ◽  
A R Henderson
Keyword(s):  
Creatine Kinase ◽  
Fluorescent Light ◽  
Human Origin ◽  
Creatine Kinase Isoenzyme ◽  
Aqueous Buffer ◽  
Creatine Kinase Isoenzymes ◽  
Total Creatine ◽  
The Stability ◽  
Sulfhydryl Compounds ◽  
Fast Freezing

Abstract We assessed the stability--at 20, 4 --20, and --80 degrees C--of 2-mercaptoethanol, dithioerythritol, and dithiothreitol, and of semi-purified creatine kinase isoenzymes of human origin in the presence and absence of the three compounds. The isoenzymes plus the sulfhydryl compounds were also assessed an aqueous buffer, in heat-inactivated pooled sera, or in fresh pooled sera with low endogenous total creatine kinase. We also examined the influence of thawing conditions from--20 or--80 degrees C, of fast freezing, and of exposure to fluorescent light. At 20 and 4 degrees C some--SH groups are oxidized. At--20 and--80 degrees C this loss is diminished. It is a function of both temperature and diluent. For stability, creative kinase isoenzymes stored at any temperature require the presence of a suitable sulfhydryl compound. Periodic addition of fresh sulfhydryl solutions stabilized creatine kinase isoenzymes suspended in the two protein-based diluents by 10-30%; for those in an aqueous buffer such additions decreased the activity to stored creatine kinase isoenzymes. Fast freezing does not increase recovery of creatine kinase isoenzyme activity over slow freezing at--80 or--20 degrees C. Thawing should be done as infrequently as possible and should be done rapidly, at 37 degrees C, for the residual enzyme activity to be maximal.

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