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Biomedical subjects

M Molinaro

Publications and source records attributed to M Molinaro.

At least 91 records · Page 5Linked to original sources
PubMed1983

Differential response of satellite cells and embryonic myoblasts to a tumor promoter.

Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) reversibly suppressed myotube formation and expression of acetylcholine receptors in cultures of Day 15 mouse embryo presumptive myoblasts, but was totally ineffective in cultures of adult mouse satellite cells. A subpopulation of TPA-resistant myogenic cells became apparent in cultures prepared from older embryos or newborn mice. Thus, limb presumptive myoblasts are a heterogeneous population, and part of the distinct TPA-resistant subpopulation may represent satellite cell precursors.

Age Factors↗
PubMed1983

Enhanced synthesis of a specific protein by 12-O-tetradecanoylphorbol-13-acetate in cultured chick embryo muscle cells.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces in cultured postmitotic myotubes specific alterations of synthesized proteins as revealed by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis after pulse labeling with [35S]methionine. Synthesis of myosin heavy chain is remarkably inhibited after exposure to 1.6 X 10(-7) M TPA for periods of 9 hr or longer. During shorter periods of TPA treatment (2 hr), an enhanced synthesis of a Mr 31,000 polypeptide is observed, which is associated with the particulate fraction of cultured myotubes. "Pulse chase" experiments show that this polypeptide is not a degradation product induced by TPA. The stimulation of Mr 31,000 polypeptide requires simultaneous RNA synthesis, since actinomycin D completely and selectively abolishes [35S]methionine incorporation into this polypeptide. The stimulation of Mr 31,000 polypeptide is a transient biosynthetic event not detectable after prolonged incubation (24 hr) of myotubes with the tumor promoter. However, TPA-containing medium preincubated with cultures for up to 24 hr induces stimulation of Mr 31,000 polypeptide when administered to untreated cultures. The early stimulatory effect on Mr 31,000 polypeptide synthesis and the late inhibitory effects on contractile protein synthesis are also observed when postmitotic, unfused myoblasts, rather than myotubes, are treated with TPA.

Animals↗
PubMed1982

Glycopeptide alterations induced by 12-O-tetradecanoyl phorbol-13-acetate in chick embryo cultured myotubes.

The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) specifically inhibits the expression of differentiative traits in cultured chick embryo myotubes, without inducing them to reenter the cell cycle. We evaluated the effect of TPA on glycoconjugate synthesis in cultured myotubes under various experimental conditions. Radioactively labelled glycoconjugates were obtained by labelling control and TPA treated cultured myotubes with radioactive monosaccharides. After chloroform-methanol extraction and extensive pronase digestion, the glycoconjugates were separated on the basis of size on Sephadex G 50 columns. A relative enrichment in larger glycopeptides was induced by TPA treatment of myotubes for 24 or 48 h. Glycopeptide groups were further analyzed by affinity chromatography on ConA Sepharose columns. A marked and reproducible decrease in the affinity of medium size glycopeptides for ConA was observed as a result of TPA treatment of cultured myotubes. These modifications are reversible upon removal of the tumor promoter from the culture medium of pretreated myotubes. The reported effects of TPA, closely resembling those occurring in transformed cells, appear to be due to structural modifications of glycopeptides whose mechanism and role in transformation and modulation of differentiation are discussed.

Animals↗
PubMed1982

TPA-induced inhibition of the expression of differentiative traits in cultured myotubes: dependence on protein synthesis.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) dramatically modifies the differentiative program of myotubes, developed in culture from chick embryo skeletal myogenic cells. In fact TPA selectively decreases the expression of differentiative parameters with a lag of 8-10 h from its administration to the cultures. We have tested whether the reported effect of TPA depends on the synthesis of specific products during the lag phase of TPA action. The data presented indicate that inhibition of protein synthesis by the use of cycloheximide prevents the appearance of TPA induced inhibition of the expression of differentiative products, such as creatine phosphokinase (CPK) activity and acetylcholine receptors (AChR). Following removal of cycloheximide and reinitiation of normal protein synthesis, the TPA induced inhibitory effect on CPK and AChR appears after a delay of about the same length as the time lag of TPA action. Our results indicate that inhibition of protein synthesis during the lag phase of TPA action prevents the effect of this tumor promoter on myotube differentiative parameters, and suggest that the expression of differentiative traits in cultured myotubes is affected by TPA via a regulatory step implying protein synthesis.

Animals↗
PubMed1982

Early alteration induced by tumor promoters on chick embryo muscle cells in culture.

When differentiated, multinucleated cultured myotubes are treated with PMA (Phorbol-12-myristate-13-acetate), they display drastic morphological alterations and undergo inhibition of the expression of differentiative traits, without being induced to reenter the cell cycle. Differentiated myotubes, obtained after cytochalasin B treatment of primary chick embryo myoblast cultures, were treated with 1.6 x 10(7) M PMA for different times, labelled with 35S-methionine and different fractions of cell extracts were analysed by SDS-PAGE followed by fluorography. The data presented here indicate that PMA treatment induces in myotubes increased synthesis of a 31.000 Mr polypeptide (31 K) within 4 hr of treatment, while the inhibition of the synthesis of contractile proteins, such as myosin and actin, occurs only after 8 hr of treatment. Morphological alterations of myotubes require longer incubation with PMA (15-20 hr). The reported effects of PMA are not induced by non tumor promotor analogs of the drug, and pulse chase experiments indicate that 31 K stimulation is not the result of increased protein degradation induced by PMA. In addition the stimulation of 31K does not occur in cultured fibroblast indicating that this is a specific early response of differentiated myogenic cells preceeding the dedifferentiative effect of this tumor promotor.

Actins↗
PubMed1981

Evaluation of blood toxicity in chronic uremia by an improved chromatographic method.

Biogel P2 gel chromatography of ultrafiltrate from chronic uremic patients leads to better resolution of the compounds present than so far reported in the literature. Reproducibility is good. Toxicity, evaluated by cell cultures, was detected in only one chromatographic fraction. Further purification of this fraction with HPLC on a RP18 column revealed two toxic fractions, to the better defined of which a polyol structure was ascribed.

Animals↗
PubMed1980

In vitro differentiation of satellite cells isolated from normal and dystrophic mammalian muscles. A comparison with embryonic myogenic cells.

Satellite cells were isolated from skeletal muscles of adult normal and dystrophic mice (C57/6J/dy strain) by sequential digestion of tissue fragments with collagenase, hyaluronidase and trypsin. These cells exhibit in culture similar behaviour to that of embryonic myoblasts, undergoing an initial duplicative period lasting about 2--3 days, followed by a shorter phase (1--2 days) of rapid cell fusion. During the duplicative phase most of the satellite cells appear round-shaped, whereas embryonic myoblasts appear typically spindle-shaped: both cell types actively incorporate [3H]thymidine. During the subsequent days of culture an increasing number of satellite cells becomes spindle-shaped; afterwards the cells contact each other and fuse into multinucleated myotubes. The majority of spindle-shaped satellite cells is unable to incorporate [3H]thymidine, thus behaving as post-mitotic cells. Concomitantly with satellite cell fusion, an increase of about 80-fold of creatine phosphokinase (CPK) specific activity is observed. Satellite cells are able to recognize co-cultured embryonic myoblasts ([3H]thymidine-labelled): hybrid myotubes containing labelled and unlabelled nuclei are formed in these experimental conditions. Satellite cells from dystrophic animals are able to differentiate in culture and do not show appreciable differences as compared to their normal counterparts. In dystrophic myotubes, however, CPK specific activity is almost twice that observed in normal myotubes. Human dystrophic satellite cells from biopsies of adult muscle cultured in similar conditions grow and fuse into multinucleated myotubes showing a behaviour identical to normal controls.

Animals↗
PubMed1978

Biosynthetic changes in myosin heavy subunit during myogenesis in culture.

In primary culture of chick embryo muscle cells myosin synthesis is detected in mononucleated cells and increased at the onset of fusion with a maximal increment of 20-fold per plate in differentiated myotube. The possibility that the myosin synthetized by duplicating myoblast could be different from that present in post-mitotic myoblast and myotube was evaluated by investigating the regulation of its synthesis and the turnover of the molecule. Following Actinomycin D treatment (0.05 microgram/ml, 8 h), myosin synthesis is partially affected (about 50% inhibition) in pre-fusion myoblast while the synthesis is more sensitive to the drug at the onset of fusion (80% inhibition). With the progress of the differentiative stage the half-life of the molecule increases from 30 h in duplicating myoblasts to 200 h in fibers. The half-life of myosin synthetized by duplicating myoblasts in the explanted embryonic muscle, is 12 h. These data show different features of myosin heavy chains related to specific stages of differentiation and suggest the possibility that modulative changes of the molecule could induce its functional maturation during myogenesis.

Animals↗