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Biomedical subjects

M Molinaro

Publications and source records attributed to M Molinaro.

At least 109 records · Page 6Linked to original sources

Parthenogenetic activation of mouse oocytes induced by inhibitors of protein synthesis.

Recently ovulated mouse oocytes at the Metaphase II stage undergo parthenogenetic activation (as indicated by the formation of pronuclei) when incubated for 6 h in the presence of cycloheximide or puromycin; the activation response increases progressively with the concentration of inhibitor. Activation is induced with concentrations of cycloheximide that depress protein synthesis by more than 70%. Pronoculear formation occurs when protein synthesis is almost totally inhibited. Incubation of oocyte in Actinomycin D failed to initiate activation. The results show that the Metaphase II oocyte of the mouse synthesizes protein factor(s) which are necessary for the maintenance of the meiotic block. Other protein(s) having opposite effects and a different rate of turnover may also participate in activation since when the oocytes are treated with a high concentration of cycloheximide (10 microgram ml-1) for varying periods of time, or with varying concentrations for a short period of time (1 h), a more complex activation response curve is obtained. Oocytes activated with cycloheximide are capable of further development, following transfer to the oviducts of pseudopregnant recipients, in a proportion similar to that of oocytes activated in other ways.

Animals↗

DNA replication during muscle cell differentiation: identification of multiple DNA-dependent DNA polymerases.

DNA-dependent DNA polymerases have been studied during chick embryo muscle differentiation in vitro. The total activity, extracted at both low and high ionic strengths, does not change throughout the differentiative process, although DNA synthesis stops at the moment of fusion. Analyses by glycerol gradient centrifugation of the extracts at low and high ionic strengths show two major DNA polymerase forms, one sedimenting at 7.5 S and another at 3-4 S. Both enzymes are present in similar amounts in duplicating myoblasts and in post-mitotic myotubes. These data suggest that the arrest of DNA synthesis which accompanies myoblast differentiation is not dependent on the disappearance or decrease of the major DNA polymerase activities described.

Animals↗

Expression of differentiative traits in the absence of cell fusion during myogenesis in culture.

Fusion of myoblasts is inhibited in cultures at low Ca++ concentration (0.44 mM); yet creatine phosphokinase and myokinase activities as well as myosin synthesis and the appearance of post-mitotic myoblasts do not significantly differ from those of control cultures (grown at 1.04 mM Ca++) which undergo cell fusion. When Ca++ concentration is increased to the control value after the second day of culture, fusion occurs very rapidly and it is not inhibited by actinomycin D or cycloheximide. Treatment with 0.06 mM bromodeoxyuridine strongly inhibits creatine phosphokinase activity and myotubes formation. The study of the kinetics of reversal of cell fusion and of creatine phosphokinase activity after removal of the analog, shows that this process is slower than the decrease of the relative content of bromodeoxyuridine incorporated into DNA. The result obtained support the following conclusions: a) the expression of the differentiative characters examined does not require cell fusion; b) the process of myotube formation seems to imply two subsequent stages consisting first of a slow maturative process, which is followed by the actual fusion of cell membranes; the former is Ca++ independent, the latter is Ca++ dependent and does not require RNA or protein synthesis.

Adenylate Kinase↗

Acetylcholine sensitivity in replicating satellite cells.

Mononucleate myogenic cells lying within skeletal muscle fiber endomysium, the so-called satellite cells (SC), isolated from adult leg muscle of normal or dystrophic mice were studied by electrophysiological techniques. Normal SC responded to iontophoretic acetylcholine (ACh) by depolarization even during the replicative phase. Acetylcholine sensitivity increased after cell fusion. Replicating SC from dystrophic muscle did not exhibit any sensitivity to ACh, and neither did embryonic myoblasts.

Acetylcholine↗

Assessment of an LC-MS method for plasma quantification of the new immunosuppressant FK778 through comparison with HPLC-UV.

FK778 is a new immunosuppressive agent, derived from the leflunomide-active metabolite A77 1726. It inhibits de novo pyrimidine nucleotide synthesis showing efficacy in the prevention and treatment of rejection in experimental transplant models. The aim of this work was to develop an HPLC-MS method to measure FK778 in plasma for pharmacokinetic studies. The equipment used for mass evaluation was an HLPC coupled to an ion trap analyzer through an electrospray source. After precipitation of plasma proteins with acetonitrile, the supernatant was injected onto an analytical RP-C18 column. Chromatographic separation was performed under isocratic conditions, using a mobile phase consisting of ammonium acetate buffer and acetonitrile (55:45. vol/vol). MS detection was performed in the negative ionization mode by monitoring the molecular ion of FK778 (m/z 307) and IS (m/z 269), using selected ion monitoring for both. However, we observed peaks corresponding to dimers, trimers, and tetramers of FK778 (m/z 637, m/z 945, m/z 1274). The HPLC-MS method was applied to pharmacokinetics in animal models showing comparable results to those obtained by an HPLC-UV assay at 290 nm. Good agreement was observed in the plasma FK778 concentration versus time curves. The rapid preparation of samples and the short run-time make this method attractive for use in clinical practice.

Alkynes↗