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In vitro studies show that cholesterol is essential to myogenesis. We have been using zebrafish to overcome the limitations of the in vitro approach and to study the sub-cellular structures and processes involved during myogenesis. We use simvastatin—a drug widely used to prevent high levels of cholesterol and cardiovascular disease—during zebrafish skeletal muscle formation. Simvastatin is an efficient inhibitor of cholesterol synthesis that has various myotoxic consequences. Here, we employed simvastatin concentrations that cause either mild or severe morphological disturbances to observe changes in the cytoskeleton (intermediate filaments and microfilaments), extracellular matrix and adhesion markers by confocal microscopy. With low-dose simvastatin treatment, laminin was almost normal, and alpha-actinin was reduced in the myofibrils. With high simvastatin doses, laminin and vinculin were reduced and appeared discontinuous along the septa, with almost no myofibrils, and small amounts of desmin accumulating close to the septa. We also analyzed sub-cellular alterations in the embryos by electron microscopy, and demonstrate changes in embryo and somite size, septa shape, and in myofibril structure. These effects could be reversed by the addition of exogenous cholesterol. These results contribute to the understanding of the mechanisms of action of simvastatin in muscle cells in particular, and in the study of myogenesis in general.

Myosin light and heavy chains from skeletal and cardiac muscles and from the electric organ of Electrophorus electricus (L.) were characterised using biochemical and immunological methods, and compared with myosin extracted from avian, reptilian, and mammalian skeletal and cardiac muscles. The results indicate that the electric tissue has a myosin light chain 1 (LC1) and a muscle-specific myosin heavy chain. We also show that monoclonal antibody F109-12A8 (against LC1 and LC2) recognizes LC1 of myosin from human skeletal and cardiac muscles as well as those of rabbit, lizard, chick, and electric eel. However, only cardiac muscles from humans and rabbits have LC2, which is recognized by antibody F109-16F4. The data presented confirm the muscle origin of the electric tissue of E. electricus. This electric tissue has a profile of LC1 protein expression that resembles the myosin from cardiac muscle of the eel more than that from eel skeletal muscle. This work raises an interesting question about the ontogenesis and differentiation of the electric tissue of E. electricus. J. Exp. Zool. 290:227–233, 2001. © 2001 Wiley-Liss, Inc.

The effects of the glycoinositolphospholipids (GIPLs) from Trypanosoma cruzion T lymphocyte activation were investigated in a mouse T cell hybridoma (DO‐11.10). Purified GIPLs from T. cruzistrains Y and G markedly increased IL‐2 mRNA transcripts and IL‐2 secretion induced by mitogenic anti‐CD3 and anti‐Thy1 mAbs. This costimulatory function was also revealed by the induction of IL‐2 secretion after the simultaneous addition of the T. cruziGIPLs and either the calcium ionophore A23187 or phorbol ester. The capacity of the GIPL molecule to induce an increase in cytoplasmic calcium levels was also demonstrated. After exposure of T cell hybridoma to GIPL, the nuclear transcription factor NFAT1 became partially dephosphory‐lated, and its nuclear localization was demonstrated both in the T cell hybridoma and in Balb/c CD3+cells. These results demonstrate that T. cruziGIPL molecules are capable of signaling to T cells and therefore could be valuable tools for the study of T cell activation, besides playing a potential role in subverting the T lymphocyte immune response during T. cruziinfection.—Bellio, M., Oliveira, A.‐C. S. C., Mermelstein, C. S., Capella, M. A. M., Viola, J. P. B., Levraud, J.‐P., Dosreis, G. A., Pre‐viato, J. O., Mendonça‐Previato, L. Costimulatory action of glycoinositolphospholipids from Trypano‐soma cruzi:increased interleukin 2 secretion and induction of nuclear translocation of the nuclear factor of activated T cells 1. FASEB J. 13, 1627–1636 (1999)

The cocarcinogenic phorbol ester 13-tetradecanoyl-O-phorbol acetate selectively and reversibly inhibits the ongoing differentiation programme of chick muscle cells in culture. 13-tetradecanoyl-O-phorbol acetate promptly blocks spontaneous contractions in mature myotubes and induces them to retract, forming giant myosacs and concurrently stress fibre-like structures are assembled. Using indirect immunofluorescence to localise desmin, the muscle specific intermediate filament protein, it was shown that its distribution is longitudinally oriented in mature myotubes. In myosacs, desmin has a reticular pattern although not as linearly oriented as in control myotubes. Using gel electrophoresis of control and 13-tetradecanoyl-O-phorbol acetate treated cell extracts, three major protein bands were observed with molecular weight of 43, 50 and 55 kDa. They migrate as actin, desmin and vimentin, respectively. The 50 kDa and 55 kDa proteins were expressed more in 13-tetradecanoyl-O-phorbol acetate-treated cells. The 50 kDa band was confirmed as desmin by immunoblotting using anti-chicken desmin antibody. Two-dimensional gel electrophoresis analysis showed the appearance of more acidic isoforms of the 50 and 55 kDa proteins 13-tetradecanoyl-O-phorbol in acetate-treated cells. The 43 kDa protein was seen as three distinct isoforms in control cells and as only two isoforms in 13-tetradecanoyl-O-phorbol acetate-treated cells.